Molecular Biology Research Products Catalog and Sourcebook for Electrophoresis

Transcription

1 Molecular Biology Research Products Catalog and Sourcebook for Electrophoresis SeaKem, NuSieve and MetaPhor Agarose PAGEr and Reliant Precast Gels GelBond Film AccuGENE Buffers FlashGel System

2 Dear Valued Lonza Customer, We are pleased to introduce the Lonza Molecular Biology Research Products Catalog. Inside, you will find all of your known and trusted Lonza research products and services, as well as many new offerings designed to advance and accelerate your research: SeaKem, NuSieve, and MetaPhor Agarose specifically engineered to optimize results by fragment size, sample type and application, including Genetic Technology Grade (GTG ) Agarose that is performance tested and certified for recovery and in-gel techniques. New! The FlashGel System providing separation, recovery and documentation of nucleic acid fragments in minutes, at the bench, without UV light or handling of hazardous solutions. This system will transform the workflow in your lab. PAGEr Precast Polyacrylamide Gels these easy-to-use protein minigels offer sharper resolution, more consistent protein transfer, and longer shelf-life than any other Tris-Glycine gels, and are compatible with your existing chamber. Latitude and Reliant Precast Agarose Gels versatile gels in multiple sizes and well formats, pre-stained and ready-to-run in your existing chambers. AccuGENE Buffers offering a broad selection of buffers for molecular biology and electrophoresis, tested and guaranteed for best performance. Look for Lonza Cell Biology Products in the Cell Discovery Research Products Catalog. The Cell Discovery catalog features your known and trusted products, such as Clonetics Primary Cells and Media; Poetics Stem Cells and Media; BioWhittaker Media and Sera; Amaxa Nucleofector Transfection Technology, and MycoAlert Mycoplasma Detection System. Purchase any of our products directly from Lonza by calling us at , or use our full e-commerce capabilities at Should you require technical information, please contact us at or visit us at scientific.support@lonza.com. Lonza is committed to providing the highest quality products and services, and to helping researchers worldwide advance and accelerate life science research. For all of your cell discovery and molecular biology needs, turn to Lonza. We appreciate your continued support and look forward to serving you. The Lonza Molecular Biology Team

3 Table of Contents Table of Contents Customer Service Terms and Conditions Trademarks and Licensing Chapter 1 Nucleic Acid Electrophoresis 1 Contents 2 Introduction 3 Agarose 4 Precast Gels for DNA and RNA 17 Markers, Stains, and Buffers 35 Gel Support Films 42 Sequencing and Mutation Detection Products 43 Nucleic Acid FAQs 70 Chapter 2 Protein Electrophoresis 45 Contents 46 Introduction 47 PAGEr Precast Gels for Protein Electrophoresis 48 PAGEr Minigel Chamber 50 Protein Markers, Buffers, and Stains 51 IsoGel Agarose and Precast Gels for IEF 56 Agarose for Protein Separation 58 ProSieve 50 Acrylamide Gel Solution 60 GelBond PAG Support Film 61 Protein Electrophoresis FAQs 72 ii iv vi Chapter 3 Electrophoresis Sourcebook 65 Preface 65 Contents 66 I. Frequently Asked Questions 70 II. Preparation of Agarose Gels 75 III. Loading and Running DNA in Agarose Gels 89 IV.Detection and Sizing of DNA in Agarose Gels 95 V. In-Gel Reactions 105 VI. Recovery of DNA from Agarose Gels 115 VII. Separation of DNA in Polyacrylamide Gels 125 VIII. Separation of RNA in Agarose Gels 131 IX. Special Applications in Agarose Gels 149 X. Protein Separation in Polyacrylamide Gels 155 XI. Blotting Proteins from Polyacrylamide Gels 171 XII. Isoelectric Focusing of Proteins on Agarose Gels 177 XIII. Protein Separation in Agarose Gels 195 Appx. A. Electrophoretic Theory 203 Appx. B. Agarose Physical Chemistry 207 Appx. C. Safety and Environmetal Precautions 210 Chapter 4 Indices 211 Product listing by Catalog Number 212 Product listing Alphabetically 215 i

4 Customer Service Customer Service U.S. and Canada Ordering Information To place an order, please use any of the following convenient options: Phone Our Customer Service Representatives are ready to assist you with ordering Monday through Friday, from 8 a.m. to 5:30 p.m. Eastern Standard Time (EST). Call from anywhere in the U.S. and Canada at: Toll Free: (800) Phone: (207) Fax Fax: (207) Mail Lonza Rockland, Inc. Customer Service Department 191 Thomaston Street Rockland, ME USA Scientific Support Providing world-class technical support for our products is a top priority. Our Scientific Support Representatives rely on years of laboratory experience to assist with product selection and help you maximize product performance. In the U.S., call us at (800) , Monday through Friday, between 8 a.m. and 5:30 p.m. EST. Outside the U.S., call (207) You may also reach us by at scientific.support@lonza.com Full product information, instructions, and MSDS are also available at Please include all of the following information on all orders: Purchase order number Shipping address and billing address Contact name and telephone number Name and department of end user Quote or reserve lot information Item number, size, quantity, and name of products ordered Online Ordering Ordering for all of our products is available 24 hours/day, 7 days per week. Our online ordering system is secure, fast, and convenient. Registered users enjoy the ability to see their contracted prices, track orders, and manage their account. Go to for more information. ii Ordering Information Outside the U.S. For Europe see page iii. For current information on internation Lonza distributors, use our website, Contact your local Lonza distributor to place your order. In the event that you do not have an authorized distributor, please contact: Lonza Rockland, Inc. Customer Service Department 191 Thomaston Street Rockland, ME USA Phone: (207) Fax: (207) Additional Lonza Research Products For information about Lonza products for Cell Biology, Transfection, Cell Culture, Gene Expression Array and BioAssay products and services, contact Lonza Walkersville, Inc. (301)

5 Customer Service International Ordering Information To place an order, please use any of the following convenient options: Phone Our Customer Service Representatives are ready to assist you with ordering Monday through Friday. International Offices Australia Austria Belgium Brazil Denmark France Germany India Italy Japan Poland Singapore Spain Sweden Switzerland The Netherlands United Kingdom Fax International Offices Ausralia Austria Belgium Brazil Denmark France Germany India Italy Japan Poland Singapore Spain Sweden Switzerland The Netherlands United Kingdom Please include all of the following information on all orders: Purchase order number Shipping address and billing address Contact name and telephone number Name and department of end user Quote or reserve lot information Item number, size, quantity, and name of products ordered Online Ordering Online ordering for all of our products is available 24 hours/day, 7 days per week. Our online ordering system is secure, fast, and convenient. Registered users enjoy the ability to see their contracted prices, track orders, and manage their account. Go to for more information. Lonza Distributors and Sales Offices For current information on Lonza distributors and sales offices, use our website, Contact your local Lonza distributor to place your order. In the event that you do not have an authorized distributor, please contact: Lonza Verviers, S.p.r.l. Verviers, Belgium Phone: Fax: info.europe@lonza.com Scientific Support Providing world-class technical support for our products is a top priority. Our Scientific Support Representatives rely on years of laboratory experience to assist with product selection and help you maximize product performance. You may reach Scientific Support by phone or Phone: scientific.support.eu@lonza.com or Full product information, instructions, and MSDS are also available at Customer Service iii

6 Terms and Conditions Terms and Conditions World Lonza Rockland, Inc. / Lonza Sales Ltd Definitions These Terms and Conditions apply to Lonza Rockland, Inc. and Lonza Sales Ltd, as the case may be, herein referred to as Seller and to all products ordered from the Lonza Research Products Catalog and Sourcebook for Electrophoresis Pricing and Terms Visa, American Express, and MasterCard credit cards are accepted. Prices and conditions are subject to change without notice. The price applicable to any order accepted by Seller shall be the price in effect on the date of shipment. Payment is due within 30 days following the invoice date. There is a minimum $50 order requirement for all orders. All claims by purchaser shall be made by written notice to Seller in accordance with these Terms and Conditions, and no offset or deduction from any invoice is permitted. Acceptance by Seller of bank draft, check, or other media of payment is subject to immediate collection of the full face amount thereof. If at any time the financial responsibility of purchaser, or the credit risk involved, shall become unsatisfactory to Seller, Seller may require cash or satisfactory security prior to subsequent shipments or deliveries hereunder. The election by Seller to require such cash or security shall not affect the obligation of purchaser to take and pay for the contracted materials. Purchaser agrees to pay all costs and expenses, including reasonable attorneys' fees, incurred by Seller in the collection of any sum payable by purchaser to Seller. If purchaser breaches any term of the Terms and Conditions or any other contractual obligation in favor of Seller, (a) Seller may choose to defer any or all further shipments or other performance of any other contractual obligation in favor of purchaser until purchaser cures its breach, and (b) Seller may, by delivery of written notice to purchaser describing the breach, immediately terminate any other contractual obligation to purchaser; provided that purchaser shall have ten (10) days after receipt of the written notice to reinstate any terminated contractual obligations by curing the breach. In the event of a termination, all outstanding payment obligations or other indebtedness of purchaser to Seller shall be due and payable no later than fifteen (15) days after delivery of notice of termination, subject to the right of reinstatement. Notwithstanding any provision in these Terms and Conditions, Seller shall have no obligation to pay any rebate, issue any credit or make any other payment of any kind to purchaser unless purchaser is fully in compliance with its payment and other obligations under the purchase and any other contractual obligation in favor of Seller. In addition, in the event that purchaser fails to make any payment when due, Seller shall have the right to offset any and all outstanding payment obligations or other indebtedness of purchaser to Seller against any outstanding payment obligations or other indebtedness that Seller or any of its affiliates may owe purchaser. Taxes In addition to the purchase price, purchaser shall pay Seller any and all governmental sales taxes of every kind that Seller may be required to pay with respect to the products. Purchaser shall provide Seller, on request, with properly completed exemption certificates for any tax from which purchaser claims exemption. Shipping All orders are shipped via Seller s recommended carriers unless an alternative agreement has been arranged with the purchaser. Applicable freight and handling charges will be added to purchaser s invoice. Refrigerated shipments will be charged a nominal fee. Please consult a Customer Service Representative for confirmed delivery dates. If another carrier is specified, a carrier account number must be provided by purchaser. Damaged or Missing Product Contact a Customer Service Representative for replacement of damaged or missing product. Damage or discrepancies must be reported within 5 business days after receipt of product. Returns Products may not be returned for credit without first obtaining authorization from a Scientific Support or Customer Service Representative. A restocking fee may be applied for products returned without authorization or for returns in which the purchaser is at fault. The restocking fee is $50 or 25% of the purchase price, whichever is greater. Force Majeure Failure of Seller to make, or purchaser to take, any one or more deliveries when due, if caused by (a) fire, storm, flood, strike, lockout, accident, act of war or terrorism, riot, civil commotion, embargo or similar circumstances, (b) any regulation, law, or restriction of any governmental department, commission, board, bureau, agency, court, or other instrumentality of any supranational organization of sovereign states, country, state, province, territory, commonwealth, municipality, or other political subdivision thereof (a "Governmental Authority"), any seizure or requisition of product by any Governmental Authority, or any compliance with a demand or request for such product for purposes of national or supranational defense, (c) inability of Seller to obtain any required raw material, energy source, equipment, labor or transportation, at prices and on terms deemed (by Seller) to be practicable, from Seller's usual sources of supply, or (d) any other cause or contingency beyond the reasonable control of that party (whether or not of the same kind or nature as the causes or contingencies above enumerated), shall not subject the party failing to perform to any liability to the other during the period such inability to make or take delivery shall exist. Quantities so affected may, at the option of either party, be eliminated from the purchase without liability, but the Terms and Conditions shall remain otherwise unaffected. In the event of Seller's inability, for any reason, to supply the quantities of product contemplated by the purchase, Seller may allocate its available supply among its purchasers, including departments and divisions of Seller, on such basis as Seller may deem fair and practical without liability to purchaser for any failure of performance that may result therefrom. Indemnification Seller will make available to purchaser a Material Safety Data Sheet (MSDS) for each product delivered to purchaser where required. The MSDS sets forth information concerning such product and describes precautions, if required, to be taken in the transportation, delivery, unloading, discharge, storage, handling and use of such product. Purchaser will familiarize itself with all information and precautions, including but not limited to such related to safety and health, contained in MSDSs or otherwise transmitted to purchaser by Seller at any time. Seller will instruct its employees, agents, contractors, customers or any third party which may be exposed to the product about such information and precautions and make available copies thereof to such parties. Purchaser assumes full liability and responsibility for compliance with the above-referenced information and precautions, and with all laws, statutes, ordinances and regulations of any Governmental Authority applicable to the processing, transportation, delivery, unloading, discharge, storage, handling, sale and use of each product including, without limitation, the Foreign Corrupt Practices Act and United States export control laws; in particular, without limiting the generality of the foregoing, purchaser shall not resell or ship to persons on the Denied Parties List or located within embargoed countries (in both cases as defined under the referenced export control laws). Purchaser further agrees to protect, defend and hold harmless Seller from and against all claims, demands, causes of action, damages, losses, liabilities, costs, expenses (including reasonable attorneys' fees), penalties, and judgments (each, a "Claim") associated with the processing, transportation, delivery, unloading, discharge, storage, handling, sale or use of any product after delivery which is (i) inconsistent with any information provided to purchaser or (ii) in violation of any applicable law, statute, ordinance or regulation of any Governmental Authority. Seller assumes no liability for failure of discharge or unloading implements or materials used by purchaser whether or not supplied by Seller. Since Seller has no control over purchaser's (or others') use, disposition, subsequent processing, admixing or reaction of Seller s products with other products, chemicals or materials, purchaser assumes the entire liability and responsibility therefor and agrees to protect, defend and hold harmless Seller from and against all Claims associated therewith including, without limiting the generality of the foregoing, Claims associated with infringement of any third party's intellectual property rights, patents on processes practiced by purchaser or patents on products made by purchaser. iv

7 Terms and Conditions World Lonza Rockland, Inc. / Lonza Sales Ltd Warranty DUE TO THE VARIOUS FACTORS AFFECTING RESEARCH TEST RESULTS, SELLER WARRANTS ONLY AND NOT FOR ANY PARTICULAR PURPOSE OF THE PURCHASES, THAT ALL PRODUCTS SOLD WILL PERFORM ACCORDING TO ESTABLISHED PRODUCT SPECIFICATIONS. PRODUCTS ARE SOLD WITH THE UNDERSTANDING THAT THE PURCHASER WILL DETERMINE IF THE PRODUCT IS SUITABLE FOR ITS APPLICATION. SELLER WILL REPLACE ANY PRODUCT, FREE OF CHARGE, THAT DOES NOT MEET SELLER S ESTABLISHED PRODUCT RELEASE SPECIFICATIONS. ANY TECHNICAL ADVICE FURNISHED OR RECOMMENDATION MADE BY SELLER OR ANY REPRESENTATIVE THEREOF CONCERNING ANY USE OR APPLICATION OF ANY PRODUCT IS BELIEVED TO BE RELIABLE BUT SELLER MAKES NO WARRANTY, EITHER EXPRESS OR IMPLIED, AS TO ITS ACCURACY OR COMPLETENESS OR OF THE RESULTS TO BE OBTAINED. NO STATEMENT IS INTENDED OR SHOULD BE CONSTRUED AS A RECOMMENDATION TO INFRINGE ANY EXISTING PATENT. WITH REGARD TO ANY PROCESSING OF ANY PRODUCT, PURCHASER ASSUMES FULL RESPONSIBILITY FOR QUALITY CONTROL, TESTING AND DETERMINATION OF SUITABILITY OF PRODUCT FOR ITS INTENDED APPLICATION OR USE. SELLER MAKES NO WARRANTY OF ANY KIND, EITHER EXPRESS OR IMPLIED, BY FACT OR LAW, OTHER THAN SELLER'S (I) OBLIGATION TO DELIVER PRODUCT COMPLYING WITH SELLER'S PUBLISHED SPECIFICATIONS (OR AS OTHERWISE REFERENCED IN THE TERMS AND CONDITIONS) AND (II) IMPLIED WARRANTIES OF TITLE, FREEDOM FROM ENCUMBRANCE, AND RIGHT TO TRANSFER SAME. SELLER MAKES NO WARRANTY OF FITNESS FOR A PARTICULAR PURPOSE, OR WARRANTY OF MERCHANTABILITY OTHER THAN AS STATED HEREIN. Product Use In general, all products listed in this catalog are For Research Use Only or For Laboratory Use Only and are not intended for use in diagnostic procedures unless otherwise noted. Such products are not to be used for diagnostic or drug purposes, or for administration to humans. Governing Law for orders made with Lonza Rockland, Inc. The purchase and Terms and Conditions shall take effect and be construed in accordance with the laws of the State of New York, USA. Governing Law for orders made with Lonza Sales Ltd The Purchase and Terms and Conditions shall take effect and be construed in accordance with the laws of Switzerland, without any reference to its conflicts of law. All rights reserved. Copyright Lonza Terms and Conditions Limitation of Liability SELLER SHALL NOT BE LIABLE FOR ANY DAMAGES OR INJURY TO PERSONS OR PROPERTY ARISING FROM THE PURCHASE OR USE OF THE PRODUCT. IN ADDITION, SELLER IS NOT LIABLE FOR THE PRODUCT AFTER THE PRODUCT EXPIRATION DATE OR FOR A PRODUCT THAT HAS BEEN MISUSED OR HAS BECOME UNUSABLE DUE TO IMPROPER STORAGE OR HANDLING BY PURCHASER. SELLER'S TOTAL LIABILITY AND PURCHASER'S EXCLUSIVE REMEDY FOR ANY CAUSE OF ACTION ASSOCIATED WITH THE PURCHASE, WHETHER BASED IN TORT, CONTRACT, STRICT LIABILITY OR ANY OTHER LEGAL THEORY IS EXPRESSLY LIMITED TO REPLACEMENT OF NONCONFORMING PRODUCT OR PAYMENT IN AN AMOUNT NOT TO EXCEED THE PURCHASE PRICE OF THE SPECIFIC PRODUCT FOR WHICH DAMAGES ARE CLAIMED, AT SELLER'S OPTION. IN NO EVENT SHALL SELLER BE LIABLE FOR ANY OTHER DAMAGES INCLUDING, WITHOUT LIMITATION, INCIDENTAL, SPECIAL, PUNITIVE OR CONSEQUENTIAL DAMAGES. PURCHASER SHALL INSPECT THE PRODUCT SUPPLIED HEREUNDER IMMEDIATELY AFTER DELIVERY. PURCHASER'S FAILURE TO GIVE NOTICE TO SELLER OF ANY CLAIM WITHIN FIVE (5) DAYS AFTER THE DATE OF DELIVERY SHALL CONSTITUTE UNQUALIFIED ACCEPTANCE OF THE PRODUCT AND A WAIVER BY PURCHASER OF ALL CLAIMS WITH RESPECT THERETO. Resale Products may be resold only by the Seller or its affiliate companies and authorized distributors. A list of authorized distributors and affiliate companies can be found in the back of this catalog or on our website, Product Label Please read the product label carefully for safety information and warnings related to product hazards. Some products may present flammable, toxic, or other hazards. More information regarding certain products can be obtained by reading the MSDS, available on request from Customer Service, Scientific Support or on the web at The absence of a warning must not be construed as an indication that the product is safe. All possible hazards may not be known at this time. Some of our products may contain materials of animal or human origin. Products should only be handled and used by qualified personnel familiar with the potential hazards and trained in laboratory procedures. The purchaser assumes all risks of use and/or handling. v

8 Trademarks The following are trademarks of Lonza Group or its affiliates Trademarks AccuGENE BioWhittaker Clonetics FlashGel Gel Slick Genetic Technology Grade GTG I.D.NA InCert IsoGel Latitude Long Ranger Lonza Group or its affiliates are owner or licensee** of the following patents. Products described herein may be covered by one or more of these United States patents, by pending patent applications, or by corresponding patents or applications in other countries. 5,055,517 5,219,923 6,365,341 5,143,646 5,641,626** 6,414,136 5,212,299** 5,836,445 6,464,850 D510,770 6,004,767 6,558,521 6,117,293 6,599,711 6,328,870 7,166,692 D511,386 D524,449 United Kingdom patent: 2,357,336 B 6,918,107 6,512,236 6,914,250 MDE MetaPhor NuSieve PAGEr ProSieve Reliant SeaPlaque SeaPrep SimplyLoad Singel TruBand Trademarks from other companies: ABI Prism, is a registered trademark of PE Corporation, Applied Biosystems Division Amplitaq and GeneAmp are registered trademarks of Roche Molecular Systems, Inc. AuroDye is a trademark of Amersham International. Biometra is a registered trademark of Biometra Biomedizinishce Analytik GmbH Bio-Rad, CHEF-DR, PROTEAN, MiniPROTEAN, Mini-Sub, Ready Gel, and Sub-Cell are registered trademarks of Bio-Rad Laboratories, Inc. Centipede, Millipede, EasyCast, Wolverine and Penguin are trademarks of Thermo Fisher Scientific, Inc. Coomassie is a trademark of Imperial Chemical Industries PLC. Dark Reader is a registered trademark of Clare Chemical Research, Inc. DH5 and DH5aF are trademarks of Life Technologies, Inc. E-Gel is a registered trademark of Ethrog, Inc. EN 3 HANCE and Teflon are trademarks of E.I. DuPont de Nemours & Co. Eppendorf is a registered trademark of Eppendorf AG Ficoll, is a registered trademark of GE Healthcare Bio-Sciences AB, Ltd. FisherBiotech is a registered trademark of Deutsche Bank, AG GelStar, GelBond and SeaKem are trademarks of FMC. Hoefer is a registered trademark of Hoefer Scientific Instruments Kimwipes is a trademark of Kimberly-Clark. LI-COR is a registered trademark of LI-COR, Inc. Nalgene is a trademark of Nalge Nunc International. Nunc is a trademark of Nunc. MultiPhor is a trademark of LKB-Produkter AB Corporation Parafilm is a trademark of American National Can Company. Pluronic is a registered trademark of Wyandotte Chemicals Corporation Polaroid is a registered trademark of Polaroid Corporation Rainbow is a registered trademark of Amersham International PLC Corporation Rubbermaid is a trademark of Rubbermaid, Inc. Schleicher & Schuell, S&S, and Turboblotter are registered trademarks of Schleicher & Schuell, G.m.b.H. SeaBlue, Novex, XCell SureLock and Multi-Mark are registered trademarks of Invitrogen Corporation Sephadex is a trademark of Amersham BioSciences. Sepharose is a registered trademark of Pharmacia Fine Chemicals, Inc. Silver Stain Plus is a trademark of BioRad. STYROFOAM is a registered trademark of Dow Chemical Corporation SYPRO and SYBR, are trademarks of Molecular Probes Corporation, Inc. Teflon is a registered trademark of E. I. Dupont De Nemours and Company Tiffen is a trademark of Tiffen Manufacturing Corporation Transwell is a registered trademark of Data Packaging Corporation Triton is a trademark of Rohm & Haas Co. Tween is a trademark of ICI Americas, Inc. Whatman is a trademark of Whatman Paper Ltd. Wratten is a registered trademark of Eastman Kodak Company Corp. Zwittergent is a trademark of Calbiochem-Novabiochem Corp. Important notice Because of numerous factors affecting results, Lonza products are sold on the understanding that purchasers will make their own tests to determine the suitability of these products for their particular purposes. The uses described in The Sourcebook are presented only to assist our customers in exploring possible applications for Lonza products. All information and data presented are believed to be accurate and reliable but are presented without responsibility on the part of Lonza. Lonza does not warrant against infringement of patents of third parties by reason of any uses made of the products in combination with other materials or in the operation of any process, and purchasers assume all risks of patent infringement by reason of any such use, combination, or operation. vi

9 Nucleic Acid Gel Electrophoresis Chapter 1

10 Nucleic Acid Gel Electrophoresis Nucleic Acid Gel Electrophoresis Nucleic Acid Gel Electrophoresis Introduction 3 Agarose 4 Precast Gels for DNA and RNA 17 Markers, Stains, and Buffers 35 Gel Support Films 42 Sequencing and Mutation Detection Products 43 2

11 Nucleic Acid Gel Electrophoresis From the very beginning to the next innovation Nucleic Acid Gel Electrophoresis Lonza is the leading innovator and world s most trusted supplier of agarose and precast gels. We are experts in protein and nucleic acid electrophoresis, bringing a strong history of innovation and reliability to your most important research. Our well known product brands set the standard in quality, purity and performance for electrophoresis. Covering the most extensive range of applications, our products are optimized for the unique requirements of your most critical molecular biology techniques. When our standard products do not completely fit your needs, inquire about our custom capabilities to find a product that does. SeaKem, NuSieve & MetaPhor Agarose FlashGel System Reliant, Latitude & PAGEr Precast Gels AccuGENE Buffers GelBond Gel Support Film 3

12 Agarose Selection Guide Agarose Selecting the best agarose for your application can minimize opportunity for error, optimize results, and even reduce cost. Lonza offers a wide range of agarose types that are specifically engineered to optimize results by fragment size, sample type and application. The selection tools below will get you started. The following pages will guide you to the right concentration, buffer and marker to use for best performance in your experiment. If you require additional support, consult our Sourcebook for Electrophoresis beginning on page 63. Choose the Agarose that is Right for You SeaKem Gold Agarose 1 kb - 10 Mb GTG Agarose and Applications SeaPlaque GTG Agarose SeaPlaque Agarose SeaKem GTG Agarose SeaKem LE Agarose NuSieve GTG Agarose 50 bp - 1 kb GTG 200 bp - 25 kb 200 bp - 25 kb 100 bp - 23 kb 100 bp - 23 kb GTG GTG NuSieve 3:1 Agarose 50 bp - 1 kb MetaPhor Agarose 20 bp bp bp 400 bp 600 bp 800 bp 1,000 bp 25,000 bp Fragment Size Range Agarose and Compatible Techniques High Gel Strength GTG Performance Certified Genetic Technology Grade 10,000,000 bp Low Melting Temperature SeaKem SeaKem SeaPlaque SeaPlaque NuSieve NuSieve MetaPhor SeaKem SeaPrep InCert I.D. NA Recovery Method LE GTG GTG 3:1 GTG Gold In-Gel Reactions ß-Agarase Phenol/Chloroform Recovery Columns Electroelution Freeze/Squeeze Blotting Southern <1 kb Southern >1 kb Northern <1 kb Northern >1 kb Specialty Applications Viral Plaque Assays Preparation of Megabase Samples PFGE Cell Culture Encapsulation & Embedding of Cells DNA Identity Testing Comet Assays 4 Our agarose is GUARANTEED DNase/RNase Free

13 SeaKem LE Agarose The standard for routine analysis HIGH GEL SeaKem LE Agarose is the ideal multipurpose, molecular biology grade agarose for any DNA or RNA application. Wide resolution range: 100 bp to 23 kb High gel strength Ideal for Southern and Northern blotting Consistent lot-to-lot performance Applications Broad range fragment separation Southern and Northern blotting PCR greater than 1 kb Immunoprecipitation techniques Baculovirus screening Colony lifts Ordering Information 1% SeaKem LE Agarose gel STRENGTH Cat. No. Size Price SeaKem LE Agarose g $ g $ g $ g $ kg $843 Larger package sizes are available upon request. Please inquire for pricing and availability. 1X TAE Buffer Agarose Storage Conditions 18 C 26 C 10 kb Recommended Lonza Ladders/Markers, see page to 1,000 bp DNA Marker 5 kb 5 kb 100 bp DNA Marker 2 kb Application Support Preparation of Agrose Gels, Section II, page 75 1 kb 1 kb 500 bp Lane 1: Hind III digest of Lambda DNA Lane 2: DNA Marker 1 to 10 kb (Lonza) Lane 3: 500 bp DNA Ladder (Lonza) SeaKem LE Agarose Final Agarose Concentration % bp- 7,000 bp 400 bp - 8,000 bp 800 bp - 9,000 bp 800 bp - 10,000 bp 0 1,000 2,000 5,000 10,000 25,000 bp 1X TBE Buffer 1X TAE Buffer See table on page79 for analytical specifications. Related Products AccuGENE Buffers 41 DNA Ladders and Markers 36 GelStar and SYBR Green Nucleic Acid Gel Stains 38 & 39 5

14 MetaPhor Agarose The highest resolution agarose available Agarose MetaPhor Agarose offers twice the resolution capability of standard agarose for PCR, STR and AmpFLP analysis. This intermediate melting temperature agarose rivals polyacrylamide and is capable of resolving DNA fragments differing in size by 2% between 20 bp and 800 bp. Rivals the resolution capability of polyacrylamide Eliminates the hazards associated with using polyacrylamide Fine separation of fragments 20 bp 800 bp Applications Small PCR analysis STR analysis RT-PCR Performance and Quality Tests DNA resolution: 4 bp resolution of DNA fragments at 200 bp and 16 bp resolution at 800 bp in TBE Buffer Gel background: Gel exhibits low background fluorescence after ethidium bromide staining DNA binding: None detected Storage Conditions 18 C 26 C Recommended Lonza Ladders/Markers, see page bp DNA Ladder 20 bp Extended Range DNA Ladder 50 to 1,000 bp DNA Marker 100 bp DNA Ladder DNA QuantLadder DNA Reverse QuantLadder Ordering Information Cat. No. Size Price MetaPhor Agarose g $ g $ g $1,287 Larger package sizes are available upon request. Please inquire for pricing and availability. Application Support Fast-Runnning protocols for High Resolution in MetaPhor Agarose Gels, page 93 MetaPhor Agarose 6 Final Agarose Concentration % bp - 130bp 50 bp bp 50 bp- 250 bp 100 bp bp 100 bp bp 150 bp bp ,000 bp 1X TBE Buffer 1X TAE Buffer See table on page79 for analytical specifications. Related Products AccuGENE Buffers 41 DNA Ladders and Markers 36 GelStar and SYBR Green Nucleic Acid Gel Stains 38 & 39

15 NuSieve 3:1 Agarose The reliable choice for PCR analysis HIGH GEL NuSieve 3:1 Agarose was the first and still is the most reliable choice for separating and resolving PCR and RT-PCR fragments. This molecular biology grade agarose produces strong, easy-to-handle gels, making it ideal for blotting of small fragments. Exceptional resolution of small fragments between 50 bp and 1 kb Superior gel strength for blotting Widely cited as the choice for PCR analysis Applications Small DNA and RNA fragment analysis Blotting of small fragments RT-PCR Genotyping Performance and Quality Tests Resolution: DNA fragments fl1,000 bp are finely resolved after electrophoresis Gel background: Gel exhibits low background fluorescence after ethidium bromide staining DNA binding: None detected Storage Conditions 18 C 26 C Recommended Lonza Ladders/Markers, see page bp DNA Ladder 50 to 1,000 bp DNA Marker 100 bp DNA Ladder DNA QuantLadder DNA Reverse QuantLadder Ordering Information NuSieve 3:1 Agarose gel for PCR products 1,353 bp 622 bp 404 bp 242 bp 238 bp 67 bp 34 bp PCR Product Primer-Dimer Primer STRENGTH Cat. No. Size Price NuSieve 3:1 Agarose g $ g $ g $1,113 Larger package sizes are available upon request. Please inquire for pricing and availability. A 550 bp sequence from lambda DNA was amplified (25 cycles) using primers and Taq DNA polymerase supplied in the GeneAmp Kit (Roche Molecular Systems). PCR products and controls were electro phoresed on a 4% NuSieve 3:1 Agarose gel in TAE Buffer at 5 V/cm for 3 hours. Lane 1, Msp I digest of pbr322 DNA (1.5 μg); lane 2, Hae III digest of øx174 DNA (1.5 μg); lane 3, no DNA control; lanes 4 9, PCR products resulting from different reaction conditions (7 μl of 100 μl reaction mixture); and lane 10, a positive control where kit template was added. Agarose Application Support Preparation of Agrose Gels, Section II, page 75 NuSieve 3:1 Agarose Final Agarose Concentration % bp- 400 bp 100 bp bp 100 bp bp 500 bp - 1,000 bp ,000 bp 1X TBE Buffer 1X TAE Buffer See table on page 79 for analytical specifications. Related Products AccuGENE Buffers 41 DNA Ladders and Markers 36 GelStar and SYBR Green Nucleic Acid Gel Stains 38 & 39 7

16 NuSieve GTG Agarose Performance certified for small fragment recovery and in-gel reactions LOW PERFORMANCE GTG Agarose NuSieve GTG Agarose provides optimal separation and resolution of PCR and RT-PCR fragments. This low melting ( 65 C) temperature agarose is easy-to-handle and can be used for cloning procedures directly from remelted agarose. Genetic Technology Grade Agarose is quality tested to certify performance. Fine resolution of small fragments between 50 bp and 1 kb Performance certified for digestion and ligation Applications Analysis and recovery of small DNA fragments In-gel PCR and In-gel ligations/transformations Performance and Quality Tests Enzymatic activity in the presence of remelted gel: T4 DNA ligase and transformation test Resolution: DNA fragments fl1,000 bp are finely resolved after electrophoresis Gel background: Gel exhibits low background fluorescence after ethidium bromide staining DNase and RNase activity: None detected DNA binding: None detected Storage Conditions 18 C 26 C Recommended Lonza Ladders/Markers, see page bp DNA Ladder 50 to 1,000 bp DNA Marker DNA QuantLadder DNA Reverse QuantLadder Application Support In-Gel Reactions, Section V, page 105 Recovery of DNA from Agarose Gels, Section VI, page 115 Ordering Information Fine resolution of low molecular weight DNA fragments in NuSieve GTG Agarose 1,353 bp 603 bp bp 194 bp 118 bp 72 bp 2% A B 3% A B MELTING 4% A B 622 bp bp 160 bp 147 bp 90 bp 67 bp 34 bp CERTIFIED Cat. No. Size Price NuSieve GTG Agarose g $ g $ g $1,222 Larger package sizes are available upon request. Please inquire for pricing and availability. -Agarase $ $431 See page 11 for more information. DNA fragments were separated in 2%, 3%, and 4% NuSieve GTG Agarose gels in 1X TBE Buffer. Lane A: Hae III digest of øx174 DNA, 0.5 μg/lane. Lane B: Msp I digest of pbr322 DNA, 0.5 μg/lane. Running conditions: 1X TBE at 5 V/cm. NuSieve GTG Agarose 8 Final Agarose Concentration % bp- 400 bp 100 bp bp 100 bp bp 500 bp - 1,000 bp ,000 bp 1X TBE Buffer 1X TAE Buffer See table on page 79 for analytical specifications. Related Products AccuGENE Buffers 41 DNA Ladders and Markers 36 GelStar and SYBR GreenNucleic Acid Gel Stains 38 & 39

17 SeaPlaque GTG Agarose Performance certified for large fragment recovery and in-gel reactions LOW PERFORMANCE GTG Confidently resolve fragments from 200 bp to 25 kb prior to PCR, cloning, digesting, or sequencing in the presence of re-melted SeaPlaque GTG Agarose, without additional purification steps. This low melting temperature ( 65 C) Genetic Technology Grade Agarose is quality tested to certify performance. Optimal separation range for DNA and RNA recovery of fragments: 200 bp to 25 kb Performance certified Applications Analysis and recovery of large DNA fragments In-gel PCR and In-gel ligations and transformations DNA and RNA digestion Performance and Quality Tests Enzymatic activity in the presence of remelted gel: T4 DNA ligase and transformation test Hind III and EcoR I restriction digestion test Fine resolution of DNA fragments 1,000 bp with low background after ethidium bromide staining DNase and RNase activity: None detected DNA binding: None detected Storage Conditions 18 C 26 C Recommended Lonza Ladders/Markers, see page bp DNA Ladder 1 to 10 kb DNA Marker DNA QuantLadder Ordering Information Resolution performance of SeaPlaque GTG Agarose 12 kb 6 kb 4 kb 2 kb 1 kb 0.5 kb 1X TAE Buffer 0.75% 1.00% 1.25% MELTING 1X TBE Buffer 0.75% 1.00% 1.25% CERTIFIED Cat. No. Size Price SeaPlaque GTG Agarose g $ g $586 Larger package sizes are available upon request. Please inquire for pricing and availability. -Agarase $ $431 See page 11 for more information. 12 kb 6 kb 4 kb 2 kb 1 kb 0.5 kb Agarose Application Support In-Gel Reactions, Section V, page 105 SeaPlaque and SeaPlaque GTG Agarose Separation of DNA markers in 0.75% to 1.25% SeaPlaque GTG Agarose gels in 1X TAE and TBE Buffers. 1 kb DNA Ladder, 1 μg/lane, DNA unheated prior to loading. The gels were cast in a 25.5 cm framing gel of 1% SeaKem GTG Agarose in a submarine chamber and run under 5 mm of buffer overlay at 5 V/cm for 3 hours, 40 minutes (TBE Buffer) and 4 hours, 30 minutes (TAE Buffer). Final Agarose Concentration % 100 bp - 3,000 bp bp - 6,000 bp 200 bp - 12,000 bp bp - 20,000 bp 0 1,000 2,000 5,000 10,000 20,000 bp 1X TBE Buffer 1X TAE Buffer See table on page 79 for analytical specifications. Related Products AccuGENE Buffers 41 DNA Ladders and Markers 36 GelStar and SYBR Green Nucleic Acid Gel Stains 38 & 39 9

18 SeaKem GTG Agarose Performance certified for large fragment recovery HIGH GEL PERFORMANCE GTG Agarose SeaKem GTG Agarose ensures reliable digestion and ligation from recovered DNA or RNA fragments from 100 bp to 23 kb. Our Genetic Technology Grade Agarose is quality tested to certify performance. Applications Best choice for DNA and RNA recovery and cloning 100 bp to 23 kb Performance and Quality Tests Restriction endonuclease digestion test: EcoR I and Hind III are tested for complete digestion of electroeluted, linearized pbr322 DNA Ligation of recovered DNA Fine resolution of DNA fragments 1,000 bp with low background after ethidium bromide staining DNase and RNase activity: None detected DNA binding: None detected Storage Conditions 18 C 26 C Recommended Lonza Ladders/Markers, see page to 1,000 bp DNA Marker 100 bp DNA Marker Ordering Information Resolution performance of SeaKem GTG Agarose 12 kb 6 kb 4 kb 2 kb 1 kb STRENGTH 1X TAE Buffer 0.75% 1.00% 1.25% CERTIFIED Cat. No. Size Price SeaKem GTG Agarose g $ g $ g $812 Larger package sizes are available upon request. Please inquire for pricing and availability. Application Support Recovery of DNA from Agarose Gels, Section VI, page kb 1 kb DNA Ladder (Lonza) 1 μg/lane, unheated Efficient Digestions After Recovery % Digest Using Hind III SeaKem GTG Agarose Final Agarose Concentration % bp- 7,000 bp 400 bp - 8,000 bp 800 bp - 9,000 bp 800 bp - 10,000 bp 0 1,000 2,000 5,000 10,000 25,000 bp 1X TBE Buffer 1X TAE Buffer 20 0 A B C D A) SeaKem GTG Agarose B) Competitor s agarose C) Competitor s agarose D) Competitor s agarose See table on page 79 for analytical specifications. Related Products 10 AccuGENE Buffers 41 DNA Ladders and Markers 36 GelStar and SYBR Green Nucleic Acid Gel Stains 38 & 39

19 SeaPlaque Agarose The original low melting temperature agarose LOW SeaPlaque Agarose is the original low melting temperature agarose and has been a staple in molecular biology labs for over 40 years. This molecular biology grade agarose produces gels with greater sieving capabilities from 200 bp to 25 kb, and with higher clarity than standard melting temperature agarose. Ideal for preparative DNA and RNA electrophoresis. Ideally suited for DNA and RNA recovery Also ideal for cloning of tissue culture cells and viral plaque assays Applications Preparative DNA and RNA electrophoresis Viral plaque assays Cell culture Storage Conditions 18 C 26 C Recommended Lonza Ladders/Markers, see page bp DNA Marker Ordering Information 1% SeaPlaque Agarose Gel 10 kb 2 kb 1 kb kb 1.3 kb 700 bp 400 bp MELTING Cat. No. Size Price SeaPlaque Agarose g $ g $436 Larger package sizes are available upon request. Please inquire for pricing and availability. Agarose Application Support Recovery of DNA from Agarose Gels, Section VI, page 115 -Agarase Recovers DNA and RNA from low melting temperature agarose 100 bp Lane 1: DNA Marker 1 to 10 kb (Lonza) Lane 2: DNA Marker 50 to 2,500 bp (Lonza) Lane 3: 500 bp DNA Ladder (Lonza) Lane 4: 100 bp Extended Range DNA Ladder (Lonza) -Agarase is an enzyme that will completely digest the poly saccharide backbone of molten agarose into alcohol soluble oligosaccharides. DNA electrophoresed in low melting temperature agarose gels can be recovered quantitatively after the gel is melted and then digested with this enzyme. Any remaining agarose oligosaccharides will not gel or interfere with subsequent DNA manipulations such as cloning, labeling, restriction digestion, or sequencing. Recovery of DNA from 1% SeaPlaque GTG Agarose with -Agarase Hind III-digested lambda DNA gel stained with ethidium bromide and the DNA bands excised. Gel digested with -Agarase, and DNA recovered by ethanol precipitation. The recovered DNA was applied to a 1% SeaKem GTG Agarose gel in 1X TBE Buffer and separated by electro phoresis. A photograph of the second gel stained with ethidium bromide is shown. Ordering Information Cat. No. Size Price -Agarase units $ units $431 Concentration -Agarase is supplied at 1,000 units/ml in a buffer consisting of 50% glycerol, 50 mm Tris/HCl, 100 mm NaCl, and 0.1% Triton X-100 at ph 7.5. Unit Definition One unit contains the amount of enzyme necessary to completely digest 200 mg of molten 1% SeaPlaque GTG Agarose gel prepared in 40 mm Bis Tris/HCl, 40 mm NaCl, 1 mm EDTA (ph 6.0) at 40 C in 1 hour. Performance and Quality Tests No detectable DNase or RNase Storage Conditions -20 C 11

20 SeaKem Gold Agarose Performance certified for rapid resolution of megabase DNA by PFGE HIGH GEL PERFORMANCE GTG Agarose SeaKem Gold Agarose is ideal for separating very large DNA fragments or doing pulsed field gel electrophoresis (PFGE). This Genetic Technology Grade Agarose is ideal for rapid resolution of megabase DNA, decreasing run times by up to 50% for PFGE. Capable of rapid separation of large DNA from 30 kb to 50 kb by horizontal electrophoresis or 50 kb to 10 Mb for PFGE Good multipurpose, high gel-strength agarose for separations 1,000 bp Specially manufactured to create a strong gel that is easy-to-handle Guaranteed DNase and RNase-free Applications Large fragment separation Pulsed field gel electrophoresis Blotting of megabase DNA Performance and Quality Tests Relative DNA mobility: 1.3 under PFGE conditions (SeaKem LE Agarose = 1.0) Restriction endonuclease digestion test: EcoR I and Hind III are tested for complete digestion of recovered DNA Ligation of recovered DNA Resolution: DNA fragments fl1,000 bp are finely resolved after electrophoresis Gel background: Gel exhibits low background fluorescence after ethidium bromide staining DNase and RNase activity: None detected DNA binding: None detected Storage Conditions 18 C 26 C Recommended Lonza Ladders/Markers, see page 36 DNA Marker 1 to 10 kb Tandem DNA Ladder 500 bp DNA Ladder DNA QuantLadder DNA Reverse QuantLadder Ordering Information 48 kb 19 kb 12 kb 6 kb 4 kb 2 kb 1 kb 0.5 kb 1X TAE Buffer 0.3% 0.3% 0.5% 0.5% Related Products STRENGTH CERTIFIED Cat. No. Size Price SeaKem Gold Agarose g $ g $552 Larger package sizes are available upon request. Please inquire for pricing and availability. Performance of SeaKem Gold Agarose for DNA 50 kb X TBE Buffer 0.3% 0.3% 0.5% 0.5% kb 19 kb 12 kb 8.6 kb 8.3 kb DNA markers separated in 0.3% and 0.5% SeaKem Gold Agarose gels in 1X TAE and TBE Buffers. Lanes 1 and 3 are 1 kb Ladders, 1 μg/lane, DNA unheated prior to loading. Lanes 2 and 4 are high molecular weight markers (8.3, 8.6, 10.1, 12.2, 15.0, 17.0, 19.4, 22.6, 24.8, 29.9, 33.5, 38.4, 48.5 kb), 0.3 μg/lane, DNA heated 10 minutes at 65 C prior to loading. Gels were cast in a 25.5 cm framing gel of 1% SeaKem GTG Agarose in a submarine chamber and run under 5 mm of buffer overlay at 1 V/cm for 16 hours (TAE Buffer), and 20 hours (TBE Buffer). 12 InCert Agarose and Megabase Standards 13 AccuGENE Buffers 41 DNA Ladders and Markers 36 See table on page 79 for analytical specifications. GelStar and SYBR Green Nucleic Acid Gel Stains 38 & 39

21 InCert Agarose and Megabase DNA Standards Used to prepare chromosomal DNA for PFGE InCert Agarose InCert Agarose is a low gelling temperature agarose, certified for use in the preparation and digestion of chromosomal DNA prior to pulsed field gel electrophoresis (PFGE). Ideal for PFGE projects Certified performance for chromosomal DNA preparation and restriction endonuclease digestion Megabase DNA Standards Our Megabase DNA Standards are specially prepared and tested chromosomal DNA standards offered in InCert Agarose gel plug format for easy handling during PFGE. Performance tested for reliable PFGE Saves time Standards are ready-to-use Ordering Information Cat. No. Size Price InCert Agarose g $ g $234 Larger package sizes are available upon request. Please inquire for pricing and availability. Cat. No. Size μg DNA/plug Price Lambda DNA Ladder 50 Kb to 873 Kb plugs 10 ± 2 $174 Saccharomyces cerevisiae DNA Standard 220 kb 1 Mb chromosomal DNA plugs 5 ± 2 $141 Performance of Megabase DNA Standards in PFGE Lambda Ladders S. cerevisiae Agarose Applications Pulsed field gel electrophoresis Performance and Quality Tests for InCert Agarose Restriction endonuclease digestion in agarose gel plugs test: enzymes tested on E. coli DNA: EcoR I, and Hind III DNase activity: None detected Storage Conditions InCert Agarose: 18 C 26 C Megabase DNA Standards: 2 C 8 C Lambda DNA Ladders and S. cerevisiae and DNA Standards were run on the Bio-Rad CHEF-DR III System. Running conditions: Lambda Ladders: 1% SeaKem GTG Agarose, 0.5X TBE, switch angle 120, 6 V/cm, ramped switch time from seconds over 22 hours. S. cerevisiae: 1% SeaKem GTG Agarose, 0.5X TBE, switch angle 120, 6 V/cm, ramped switch time from seconds over 24 hours. Block 1 Block 2 Switch angle: V/cm: 2 V/cm 2 V/cm Switch time (sec.): Run time (hr.): Related Products See table on page 79 for analytical specifications. AccuGENE Buffers 41 13

22 SeaKem ME Agarose Ideal for serum protein and IEP analysis Agarose SeaKem ME Agarose is the ideal choice for serum protein electrophoresis and immunoelectrophoresis, and may be used for DNA electrophoresis. Enhanced resolution in serum protein electrophoresis High gel clarity and minimal non-specific binding Applications Serum protein electrophoresis Immunoelectrophoresis Nucleic acid electrophoresis Storage Conditions 18 C 26 C Ordering Information Cat. No. Size Price SeaKem ME Agarose g $ g $ g $627 Larger package sizes are available upon request. Please inquire for pricing and availability. Application Support Protein Separation in Agarose gels, Section XIII, page 195 SeaPrep Agarose Ideal for cell culture applications SeaPrep Agarose is a unique ultra-soft agarose, ideal for high e ciency hybridoma cloning. It is also used for expanding cdna libraries in a strictly representative fashion, decreasing the possibility that less abundant clones vanish during amplification due to differential rates of replication. Specifications Melting temp: 50 1% Gelling temp: 8 C % Gel Strength: >75 Applications Cell culture Hybridoma cloning Encapsulation/embedding of cells Storage Conditions 18 C 26 C Ordering Information Cat. No. Size Price SeaPrep Agarose g $210 Application Support Amplification of cdna libraries with SeaPrep Agarose, Section IX, page 150 Preparing Agarose for use in Cell Culture Applications, Section IX, page 152 Related Products 14 See table on page 79 for analytical specifications. AccuGENE Buffers 41

23 I.D.NA Agarose Designed for identity testing I.D.fifi Agarose is specially manufactured for DNA identity testing. For reliable separation of VNTRs, HVRs, RFLPs, and DNA size standards, it is a perfect match for your DNA typing tests. Performance certified to assure lot-to-lot reliability for DNA identity testing Crisp DNA separation to accurately discriminate DNA fragments Strong, easy-to-handle gels allow for trouble-free high efi ciency blotting Applications DNA identity testing Performance and Quality Tests DNase and RNase activity: None detected DNA binding: None detected Storage Conditions 18 C 26 C Ordering Information Cat. No. Size Price I.D.NA Agarose g $279 Larger package sizes are available upon request. Please inquire for pricing and availability. Resolution and transfer performance of I.D.NA Agarose kb 10 kb 5.2 kb Agarose 2.3 kb 1.3 kb An autoradiogram of DNA sizestandards (LIFECODES Corp.) and Hae IIIdigested K562 DNA probed with D4S139 (Invitrogen). DNA was electrophoresed at 1 V/cm for 16 hours in a 1% I.D. Agarose gel, transferred, and probed. Lane 1: DNA size standards; Lane 2: alleles detected with D4S139. Related Products AccuGENE Buffers 41 DNA Ladders and Markers 36 GelStar and SYBR Nucleic Acid Gel Stains 38 & 39 Long Ranger Singel Packs 43 MetaPhor Agarose 6 15

24 Notes 16

25 Precast Gels for DNA and RNA Selection Guide Lonza offers a complete family of precast agarose gels for DNA and RNA electrophoresis. Our unique gel options cover the full range of separations needs, from ultra-fast PCR analysis and recovery, to fine resolution and high-throughput separations. Our custom manufacturing capabilities can support the requirements of nearly any application. All Lonza gels are precision manufactured with our high quality SeaKem and NuSieve Agarose and functionally tested for consistent performance. FlashGel System Reliant Gel Minigels No. of Wells , and Loading Volume 5 μl, 12 μl 8.4 cm Five minute DNA separation: Separate DNA 10 bp to 10 kb and RNA 0.5 kb to 9 kb Watch DNA migrate in real time, without UV light Recover samples directly, without purification See pages for a complete product description 7.0 cm No. of Wells 20 Loading Volume <15 μl 9.5 cm Small format gels for DNA and RNA: Run 8 24 samples Ideal for blotting and recovery 6 cm Fits standard horizontal chambers No. of Wells 8, 12 and 24 Loading Volume <15 μl 6 cm 9.5 cm Precast Gels Latitude HT Gels See pages for a complete product description Large format gels for DNA: Run samples PAGEr Gold TBE Gels No. of Wells 100 and 200 Loading Volume 10 μl 12 μl 25 μl 30 μl 24 cm Ideal for high throughput screening of DNA samples Fits standard horizontal chambers See page 28 for a complete product description 14 cm Latitude Midigels No. of Wells 20 and 40 Loading Volume 10 μl 12 μl 10 cm 15 cm Medium format gels for DNA and RNA: Run samples Ideal for routine analysis, blotting and recovery Fits standard horizontal chambers See page 30 for a complete product description No. of Loading Wells Volume μl μl μl 9 cm No. of Loading Wells Volume μl μl μl 10 cm 10 cm Vertical polyacrylamide gels for DNA: Run samples Ideal for fine resolution Easy to load and open 10 cm See page 31 for a complete product description 17

26 FlashGel System Fast, sensitive, simple analysis, recovery, and documentation of DNA and RNA Precast Gels FlashGel System The FlashGel System provides nearly instant results. Simply load samples, watch bands migrate and get data in as little as 2 minutes. Now the FlashGel System has expanded to include DNA recovery and gel documentation. Say goodbye to gel preparation, band excision, purification, and UV light. Complete separation, recovery and documentation safely, at the bench, in minutes. 5 minute separation and recovery See bands in as little as 2 minutes Recover samples directly, without UV light, band excision or purification Real-time separation and documentation Watch band migration as it happens Photograph gels at the bench, without DNA damaging UV light Outstanding sensitivity and resolution 520 times more sensitive than EtBr; detect < 0.1 ng DNA or < 10 ng total RNA Clean, sharp separation and straight, uniform sample lanes The FlashGel System consists of enclosed, disposable, precast agarose gel cassettes and a combination electrophoresis and transilluminator unit. FlashGel Cassettes contain precast, prestained agarose gels and buffer no need for gel preparation, buffer addition or gel staining The FlashGel Dock is an electrophoresis apparatus with a built-in transilluminator that provides both separation and detection The FlashGel Camera is a compact camera system designed to photograph FlashGel Cassettes right at the bench. FlashGel Markers are recommended for best performance FlashGel System FlashGel System for DNA 5 minute DNA analysis The FlashGel System for DNA is the ideal sample screening tool. Check up to 34 PCR or restriction fragments quickly, without having to plan your day around agarose gels. Fast, simple procedure Separation at various run times on the FlashGel System 2 minutes 4 minutes 6 minutes 8 minutes Insert cassette into dock. Pre-load wells with distilled or deionized water. Load samples. Plug in and turn on light and electrophoresis voltage. Watch until desired separation is achieved. Photograph. 1 kb 500 bp 100 bp Markers run on a 1.2% FlashGel Cassette, 12+1 well format, 275V for times as shown. Sample lanes from left to right: FlashGel DNA Marker (100 bp 4 Kb), FlashGel QuantLadder, Lonza bp Marker, Lonza 100 bp Ladder. 18 See page 25 for ordering information.

27 FlashGel System for DNA 5 minute DNA analysis 5 minute separation The FlashGel System provides high voltage separation of fragments (275V for 2 7 minutes); DNA separates in a fraction of the time required by competitor precast gel systems Real-time visualization Built-in illumination, allows you to view DNA under ambient light as it migrates through the gel; stop the run when desired separation is reached (in as little as 2 minutes depending upon fragment of interest); safely view the cassette on the lighted dock without eye protection. DNA bands separated on FlashGel Cassettes are also detectable by UV light and may be photographed using standard gel documentation systems. Use The FlashGel Camera for best performance. Superior resolution Resolve fragments in 2 7 minutes, and see clean, sharp band separation, and straight, uniform sample lanes Comparison of FlashGel System with Company I % FlashGel Cassette, 12+1 well, single-tier format. 275V, 7 min run on The FlashGel Dock. Company I 1.2% gel, 12 well, single-tier format. 30 minute run. Lanes 1 and 7: FlashGel DNA Marker (100 bp 4 Kb); Lanes 2 and 8: FlashGel QuantLadder; Lanes 3 and 9: Lonza 100 bp Ladder; Lanes 4 and 10: Lonza bp Marker; Lanes 5 and 11: 285 bp -actin PCR; Lanes 6 and 12: 294 bp control PCR (Company A) Samples diluted with 1X FlashGel Loading Dye prior to loading. Dilutions and load volumes optimized for each sample in each gel system. Precast Gels FlashGel System DNA bands as viewed during a run on the FlashGel Dock. Cassette Specifications Optimal separation range: 1.2% agarose: 50 bp 4,000 bp 2.2% agarose: 10 bp 1,000 bp Separation of fragments >4 kb will be improved by running longer at lower voltage Storage: Room temperature for 5 months from date of manufacture. Shelf life may be extended with refrigerated storage. Well volume: 12+1 well: 5 μl 16+1 well: 5 μl 8+1 well: 12 μl Gel size: 70 mm (L) X 84 mm (W) X 2 mm (H) Cassette size: 115 mm (L) X 107 mm (W) X 17 mm (H) Dock size: 134 mm (L) X 120 mm (W) X 54 mm (H) See page 25 for ordering information. Some components and technology of the FlashGel System are sold under licensing agreements. The nucleic acid stain in this product is manufactured and sold under license from Molecular Probes, Inc., and the FlashGel Cassette is sold under license from Invitrogen IP Holdings, Inc, and is for use only in research applications or quality control, and is covered by pending and issued patents. The FlashGel Dock technology contains Clare Chemical Research, Inc. Dark Reader transilluminator technology and is covered under US Patents 6,198,107; 6,512,236; and 6,914,250. The electrophoresis technology is licensed from Temple University and is covered under US Patent 6,905,585. Related Products FlashGel Dock and Cassettes FlashGel System for Recovery 20 FlashGel System for RNA 22 FlashGel Camera 24 19

28 FlashGel System for Recovery 5 minute DNA recovery Precast Gels FlashGel System Direct DNA recovery using the FlashGel System for Recovery eliminates agarose gel preparation, band excision, and purification. The system delivers highly efi cient recovery, free from inhibitors and UV-induced damage, in a simple 5-10 minute protocol. Go from sample loading to recovery in just 5 minutes Recover samples directly from the cassette, without band excision or purification Visualize sample recovery without UV Recover at 80% - 100% efi ciency Fast, simple procedure Load samples in top tier of wells. Run until samples reach the second tier of wells. Stop the run and add FlashGel Recovery Buffer. Remove DNA from wells. No DNA damaging UV or mutagenic stain exposure Visible light from the compact FlashGel Dock illuminates the recovery wells without damage to the DNA or hazard to the user The proprietary stain in the FlashGel Cassettes enables separation and recovery of very small quantities of DNA, and minimizes user exposure to potential mutagens Efficienct recovery, free from inhibitors Samples are recovered at 80% - 100% efi ciency, are free of inhibitors, and ready for subsequent re-amplification, cloning, or other techniques, without additional clean-up steps Recovery Efficiency on the FlashGel System for Recovery 1. Pre-Recovery The FlashGel Recovery System eliminates the need to cut away and then purify bands. As DNA migrates to the second tier of wells, it is free from the agarose matrix and easily extracted via pipette, with the aid of the FlashGel Recovery Buffer. Depending upon initial separation time in the recovery step, the same cassette may be used for analysis verification of the recovered sample. 2. Post-Recovery 3. Recovered Samples 2-fold dilutions of 1000 bp fragments were separated and recovered using the FlashGel Recovery System. DNA concentrations ranged from ng 600 ng. Images 1 and 2 show the cassette before and after recovery. Image 3 shows analysis of 5% of each recovered sample, run on a 1.2% FlashGel DNA Cassette. Comparison to the FlashGel QuantLadder show recovery efi ciencies ranging from 80% to 90%. 20

29 FlashGel System for Recovery 5 minute DNA recovery DNA Size Range on the FlashGel System for Recovery FlashGel Recovery System Column Recovery Method Separate fragments on FlashGel System 3-5 minutes Separate fragments on agarose gel minutes Samples were separated and recovered on a FlashGel Recovery Cassette. 3 μl aliquots of recovered samples consisting of 100 ng of fragments ranging from 50 bp to 4000 bp separated on a 1.2% FlashGel DNA Cassette and compared to the FlashGel DNA Marker 100 bp 4 kb and the FlashGel QuantLadder. Recover DNA directly from wells 1-5 minutes Total time: 4-10 minutes Excise bands from agarose gel 5-10 minutes Recover DNA via spin column minutes Total time: > 1 hour Precast Gels FlashGel System Recovery Efficiency on the FlashGel System for Recovery Cassette Specifications 1. Recovered Samples 2. Plasmid Restriction Digests FG C FG1 FG2 C1 C2 V Plasmid DNA (pbr322) was subjected to restriction enzyme double digestion using PstI and BamHI. Samples of the restricted DNA were separated and 3.2 kb fragments were recovered using the FlashGel Recovery System (FG) or spin column kits (C1 and C2). Image 1 compares 5% of each recovered sample. Aliquots of the recovered samples were ligated into PstI/BamHI double digested puc19 vector (V). Samples of the ligation reactions were transformed into E.coli competent cells. The number of colonies obtained with both samples were very similar. Image 2 shows examples of PstI/BamHI cut plasmid samples from two colonies from each sample. V shows a restricted sample of vector with no insert. Optimal separation range: 1.2% agarose: 50 bp 4,000 bp 2.2% agarose: 10 bp 1,000 bp Separation of fragments >4 kb will be improved by running longer at lower voltage Storage: Room temperature for 5 months from date of manufacture. Shelf life may be extended with refrigerated storage. Well volume: 12+1 well: 5 μl 16+1 well: 5 μl 8+1 well: 12 μl Gel size: 70 mm (L) X 84 mm (W) X 2 mm (H) Cassette size: 115 mm (L) X 107 mm (W) X 17 mm (H) Dock size: 134 mm (L) X 120 mm (W) X 54 mm (H) See page 25 for ordering information. Some components and technology of the FlashGel System are sold under licensing agreements. The nucleic acid stain in this product is manufactured and sold under license from Molecular Probes, Inc., and the FlashGel Cassette is sold under license from Invitrogen IP Holdings, Inc, and is for use only in research applications or quality control, and is covered by pending and issued patents. The FlashGel Dock technology contains Clare Chemical Research, Inc. Dark Reader transilluminator technology and is covered under US Patents 6,198,107; 6,512,236; and 6,914,250. The electrophoresis technology is licensed from Temple University and is covered under US Patent 6,905,585. Related Products FlashGel System for DNA 18 FlashGel System for RNA 22 FlashGel Camera 24 21

30 FlashGel System for RNA Rapid, sensitive, convenient RNA analysis The FlashGel System for RNA is optimized for the unique requirements of RNA, and is the ideal tool for rapid analysis of sample integrity. High quality, intact RNA is essential for consistent results in gene expression, Northern analysis, cdna library construction and cdna labeling for microarrays. Most protocols recommend checking RNA prior to downstream analysis. Separation of total RNA on the FlashGel System for RNA Precast Gels FlashGel System The FlashGel System completes RNA analysis in less than 30 minutes and requires < 10 ng total RNA for detection. The system is recommended for verification and analysis of total RNA, quick checks of native RNA and checking for RNA degradation and mrna purity. Get results in 30 minutes or less Detect < 10 ng RNA per band Avoid hazardous reagents and contaminating RNases Rapid RNA Analysis Samples of RNA Marker (Lonza) (lanes 1 and 2) and E. coli total RNA (lanes 3 and 4) contain 50 ng (lanes 1 and 3) or 250 ng (lanes 2 and 4) of RNA per 5 μl load. Samples prepared with Formaldehyde sample buffer (Lonza) and denatured 5 minutes at 65 C. FlashGel RNA Cassette run for 8 minutes, followed by a 20 minute hold prior to imaging Insert cassette into FlashGel Dock. Pre-load wells with RNase-free water. Load samples. Plug in and turn on light and electrophoresis voltage. Run for 8 minutes. Turn off voltage and hold for 10 minutes, or until RNA bands are stained to the desired intensity. Photograph. Checking sample quality with the FlashGel System for RNA Exquisitely sensitive detection The FlashGel System for RNA offers the detection sensitivity of a chip system, without the cost, and rivals the best RNA stains (SYBR Green and GelStar Stains), without direct handling of stain solutions. RNA quantities < 10 ng per band are clearly detected on the FlashGel System, conserving precious RNA samples Clean, enclosed system FlashGel RNA Cassettes fully enclose the gel, stain and running buffer, eliminating user exposure to hazardous reagents, and protecting samples from contaminating RNases. RNA cassettes are designed for performance and purity, and are guaranteed RNase free. The FlashGel Dock provides electrophoresis and visual ization of both DNA and RNA cassettes Sample degradation is visible at low levels on the FlashGel System for RNA. FlashGel RNA Cassette run for 8 minutes at 225 V, followed by 20 minute hold prior to imaging. Lane 1: RNA Marker (Lonza); Lane 2: 250 ng E. coli Total RNA; Lanes 3 7: E. coli Total RNA incubated with increasing levels of RNase A. Intact, denatured RNA shows sharp, clear bands on the FlashGel System. Partially degraded RNA has a smeared appearance, and completely degraded RNA appears as a low molecular weight smear. FlashGel Dock and Cassettes 22

31 FlashGel System for RNA Rapid, sensitive, convenient RNA analysis Quick check of native RNA on the FlashGel System for RNA FlashGel RNA Cassette run for 4 minutes at 225 V, followed by immediate imaging. Lanes 1 4: native samples prepared with FlashGel Loading Dye. Lanes 5 8: samples denatured with formaldehyde at 65 C for 5 minutes prior to loading. Lanes 1, 2, 5 and 6 contain a 1.25 μl load. Lanes 3, 4, 7 and 8 contain a 5 μl load. Specifications Separation range: Storage: Well volume: Gel size: Cassette size: Dock size: See page 25 for ordering information. 0.5 kb 9 kb Room temperature for 3 months from date of manufacture. Shelf life may be extended with refrigerated storage well: 5 μl 16+1 well: 5 μl 70 mm (L) X 84 mm (W) X 2 mm (H) 115 mm (L) X 107 mm (W) X 17 mm (H) 134 mm (L) X 120 mm (W) X 54 mm (H) Precast Gels FlashGel System Comparison of Agarose Gels for RNA A B C D Panel Gel Sample preparation Run conditions Total time A B C D FlashGel System for RNA Reliant 1.25% MOPS Gel (Cat. #54922) Reliant 1.25% MOPS Gel Hand-cast 1% SeaKem LE Agarose Gel (Cat. #50002) Formaldehyde Sample Buffer (Cat.# 50571) Samples denatured 5 Min. at 65 C. 10μg/ml EtBr added to Formaldehyde Sample Buffer. Samples denatured 5 Min. at 65 C. 8 min run at 225 V. Cassette held at ambient temp for 20 min prior to imaging. 60 min run at 5 V/cm. 60 min post-stain with GelStar Stain (Cat. #50535) 1:5000 dilution 60 min run at 5 V/cm. 60 min EtBr post-stain and 60 min de-stain. Denaturing gel prepared at 2.2 M Formaldehyde in 1X MOPS buffer (Cat. #50876). 60 min run at 6 V/cm. 28 min 120 min 108 min 120 min Some components and technology of the FlashGel System are sold under licensing agreements. The nucleic acid stain in this product is manufactured and sold under license from Molecular Probes, Inc., and the FlashGel Cassette is sold under license from Invitrogen IP Holdings, Inc, and is for use only in research applications or quality control, and is covered by pending and issued patents. The FlashGel Dock technology contains Clare Chemical Research, Inc. Dark Reader Transilluminator technology and is covered under US Patents 6,198,107; 6,512,236; and 6,914,250. The electrophoresis technology is licensed from Temple University and is covered under US Patent 6,905,585. Performance of RNA analysis in four agarose gel systems. Sample of RNA Marker (Cat. #50575) lanes 1 & 2, and E.coli Total RNA (Ambion) Lanes 3 & 4 contain 50 ng (lanes 1 & 3 or 250 ng (lanes 2 & 4) of RNA per 5 μl load. Related Products FlashGel System for Recovery 20 FlashGel System for DNA 18 FlashGel Camera 24 23

32 FlashGel Camera From benchtop to desktop in 5 minutes Precast Gels FlashGel System Capture data from The FlashGel System and say goodbye to darkrooms and UV light. Complete separation and documentation safely, at your bench in minutes. This simple digital camera in an enclosed hood connects directly to your laptop or PC via USB. Now you can visualize band migration on The FlashGel Dock or via image projected to your computer screen. Simply click a button to capture the desired image to a file. Real time separation and documentation Complete your gel run and image capture in just 5 minutes Watch DNA migrate in the FlashGel Dock or on your computer screen Photograph gels at the bench without UV light The FlashGel Camera offers Sharp, clear high-resolution images Simple user interface Small, compact design Optimized exposure for FlashGel Cassettes FlashGel Camera Camera Specifications Hood dimensions: 10 cm (W) x 11 cm (L) x 16 cm (H) Camera type: Digital Image file type:.jpg,.tif,.pdf See page 25 for ordering information. Simple user interface right from your laptop or PC Related Products 24 FlashGel System 18

33 FlashGel System Ordering Information Purchase components of the FlashGel System separately, as a complete system or as starter kits for DNA, RNA or recovery applications. Ordering Information Cat. No. Description Price FlashGel Dock Each $399 For use with all FlashGel Cassette types FlashGel Camera Each $545 Includes: Camera, hood enclosure USB cable and FlashGel Capture software. For use with FlashGel Dock. FlashGel System Each $990 Includes: FlashGel Dock; FlashGel Camera; 9 pk FlashGel DNA Cassettes (1.2%, 12+1-well single-tier); FlashGel Loading Dye and DNA Marker. FlashGel System for DNA Ordering Information Cat. No. Description/Size Price FlashGel DNA Cassettes 9/pk % agarose, 12+1 single-tier $ % agarose, 16+1 double-tier $ % agarose, 12+1 single-tier $ % agarose, 16+1 double-tier $99 FlashGel Loading Dye (5X) (contains xylene cyanol) ml $126 FlashGel DNA Marker, 50 bp 1.5 kb Ready-to-Load (Recommended for 2.2% cassettes), 50 applications μl $114 FlashGel DNA Marker, 100 bp 3 kb Ready-to-Load (Recommended for double-tier cassettes), 50 applications μl $114 FlashGel DNA Marker, 100 bp 4 kb Ready-to-Load (Recommended for 1.2% cassettes), 50 applications μl $114 FlashGel QuantLadder, 100 bp (3 ng) 1.5 kb (30 ng) Ready-to-Load, 50 applications μl $114 FlashGel DNA Starter Kit Includes FlashGel Dock; FlashGel Loading Dye; FlashGel DNA Cassettes, 1.2%, 12+1 well single-tier, 9 pk; FlashGel DNA Marker 100 bp 4 kb Each $497 See page 97 for detailed marker sizes. FlashGel System for Recovery Ordering Information Cat. No. Description/Size Price FlashGel Recovery Cassettes 9/pk % agarose, 8+1 double-tier Inquire* FlashGel Recovery Buffer Ready-to-Use x 500 μl Inquire* FlashGel QuantLadder 100 bp (3 ng) 1.5 kb (30 ng) Ready-to-Load, 50 applications μl $114 FlashGel Recovery Starter Kit Includes FlashGel Recovery Cassettes, 1.2%, 8+1 well double-tier, 9 pk; FlashGel Loading Dye; FlashGel Recovery Buffer; FlashGel QuantLadder; Visualization Glasses; Control Fragment. Dock sold Separately Each Inquire* *Avaiable soon. FlashGel System for RNA Ordering Information Cat. No. Description/Size Price FlashGel RNA Cassettes 9/pk % agarose, 12+1 single-tier $ % agarose, 16+1 double-tier $99 Formaldehyde Sample Buffer RNA denaturing sample buffer(contains bromophenol blue and xylene cyanol) ml $39 FlashGel Loading Dye For native RNA samples(contains xylene cyanol) ml, 5X concentrate $126 FlashGel RNA Marker, 0.5 kb 9 kb μg (1 μg/ml) $136 AccuGENE Molecular Biology Water RNase Free L $26 FlashGel System for RNA Starter Pack Includes FlashGel RNA Cassettes1.2%, well, Single-tier, 9 pk; Sample Buffer, RNA Marker, and Molecular Biology Water. Dock sold separately Each $264 NOTE: Due to varying storage requirements, kit components may arrive in separate shipping containers. Precast Gels FlashGel System 25

34 Reliant Minigels Versatile minigels for routine DNA separation and recovery Reliant Gels are versatile and convenient minigels for nearly any application. Each gel is precision manufactured for rapid and reproducible resolution of DNA sizes from 8 bp to 10 kb. Reliant Gels are available in a variety of well formats and agarose concentrations, in TAE and TBE Buffer and most are prestained with ethidium bromide. Manufactured with high quality SeaKem and NuSieve Agarose for reliability Compatible with most minigel chambers Versatile format options Precast Gels 6 cm 6 cm 9.5 cm 9.5 cm 9.5 cm Ordering Information Cat. No. Stain Buffer Separation Range (bp) Agarose Price Reliant Gel 8 well 20 gels/box No Stain TAE 400 fi 10,000 1% SeaKem Gold Agarose $ Ethidium bromide (0.5 μg/ml) TAE 400 fi 10,000 1% SeaKem Gold Agarose $ Ethidium bromide (0.5 μg/ml) TAE 20 fi 1,000 4% NuSieve 3:1 Plus Agarose $ Ethidium bromide (0.5 μg/ml) TBE 300 fi 8,000 1% SeaKem Gold Agarose $ Ethidium bromide (0.5 μg/ml) TBE 8 fi 1,000 4% NuSieve 3:1 Plus Agarose $121 Reliant Gel 12 well 20 gels/box Ethidium bromide (0.5 μg/ml) TAE 400 fi 10,000 1% SeaKem Gold Agarose $ Ethidium bromide (0.5 μg/ml) TBE 300 fi 8,000 1% SeaKem Gold Agarose $ Ethidium bromide (0.5 μg/ml) TBE 100 fi 3,000 2% SeaKem Gold Agarose $ Ethidium bromide (0.5 μg/ml) TBE 8 fi 1,000 4% NuSieve 3:1 Plus Agarose $121 Reliant Gel 24 well 20 gels/box Ethidium bromide (0.5 μg/ml) TBE 300 fi 8,000 1% SeaKem Gold Agarose $ Ethidium bromide (0.5 μg/ml) TBE 100 fi 3,000 2% SeaKem Gold Agarose $ Ethidium bromide (0.5 μg/ml) TBE 8 fi 1,000 4% NuSieve 3:1 Plus Agarose $121 6 cm 6 cm Reliant Gel 20 well 20 gels/box No Stain TBE 100 fi 3,000 2% SeaKem Gold Agarose $ No Stain TBE 8 fi 1,000 4% NuSieve 3:1 Plus Agarose $ Ethidium bromide (0.5 μg/ml) TBE 300 fi 8,000 1% SeaKem Gold Agarose $ Ethidium bromide (0.5 μg/ml) TBE 100 fi 3,000 2% SeaKem Gold Agarose $ Ethidium bromide (0.5 μg/ml) TBE 8 fi 1,000 4% NuSieve 3:1 Plus Agarose $ cm Visit or contact Scientific Support to inquire about custom precast gels. Specifications Gels per box: 20 Gel dimensions: 6.0 cm 9.5 cm Gel thickness: 5.5 mm Tray dimensions: 6.8 cm 10.2 cm Well volume: <15 μl 26

35 Reliant Minigels Continued Performance of Reliant Minigels Panel A 1X TBE + EtBr Panel B 1X TAE + EtBr Reliant Minigels Supporting Products Ordering Information Cat. No. Description Price Reliant Gel System Reusable UV Transparent Tray Landscape tray $ Portrait tray $23 Panel A. 20 bp Ladder (1 μl), 100 bp Ladder (1 μl) and bp Marker (2.5 μl) (all Lonza), loaded and run in a 4% NuSieve 3:1 Plus Reliant Gel containing ethidium bromide. Gel was run at 7 V/cm for 50 minutes using 1X TBE Buffer containing 0.5 μg/ml ethidium bromide. Panel B. A repeating pattern of 500 bp DNA Ladder (1 μl/lane) and 1-10 kb DNA Marker (2.5 μl/lane) (Lonza) run in a 1% SeaKem Gold Reliant Gel containing ethidium bromide. Gel was run at 5 V/cm for 60 minutes using 1X TAE Buffer containing 0.5 μg/ml ethidium bromide. Applications DNA analysis Restriction digests Recovery PCR RT-PCR Cloning Blotting Performance and Quality Tests DNase: No activity detected Gel performance: Sharp bands and low background fluorescence Storage Conditions 18ºC 26ºC for 6 12 months depending upon agarose concentration DNA Loading Buffer (6X) (contains bromophenol blue and xylene cyanol) ml $30 AccuGENE 50X TAE Buffer L $67 AccuGENE 10X TAE Buffer L $ L $87 AccuGENE 10X TBE Buffer L $ L $ L $ L $234 AccuGENE 5X TBE Buffer L $ L $ L $152 Precast Gels Application Support Loading and Running DNA in Agarose Gels, Section III, page 89 Detection and Sizing of DNA in Agarose Gels, Section IV, page 95 Related Products DNA Ladders and Markers 36 GelStar and SYBR Green Nucleic Acid Gel Stains 38 & 39 27

36 Latitude HT Gels Precast gels for high throughput separations Latitude HT Precast Agarose Gels are large format agarose gels designed for high throughput screening applications. These gels are precision manufactured for rapid, reproducible resolution of DNA sizes from 8 bp to 10 kb. Latitude HT Gels are available in multiple well formats (from wells) and agarose concentrations, in TAE and TBE Buffer, all prestained with ethidium bromide. Manufactured with high quality SeaKem or NuSieve Agarose for reliability Versatile design allows you to run gels in most large submerged electrophoresis systems Multichannel pipette compatible Precast Gels 24 cm 24 cm 24 cm 14 cm 14 cm 14 cm Ordering Information For optimal performance, use the TruBand Anchor with Latitude HT Gels, see page 29. Cat. No. Stain Buffer Separation Range (bp) Agarose Price Latitude HT Precast Gels 100 Well (2 50 wells) Alternate well multichannel pipette compatible, 5 gels/box Ethidium bromide (0.5 μg/ml) TAE 400 fi 10,000 1% SeaKem LE Plus Agarose $ Ethidium bromide (0.5 μg/ml) TAE 100 fi 3,000 2% SeaKem LE Plus Agarose $ Ethidium bromide (0.5 μg/ml) TBE 300 fi 8,000 1% SeaKem LE Plus Agarose $ Ethidium bromide (0.5 μg/ml) TBE 8 fi 1,000 4% NuSieve 3:1 Plus Agarose $ Ethidium bromide (0.5 μg/ml) TBE 100 fi 2,000 2% SeaKem LE Plus Agarose $132 Latitude HT Precast Gel 100 Well (4 25 wells) Consecutive well multichannel pipette compatible, 5 gels/box Ethidium bromide (0.5 μg/ml) TBE 100 fi 2,000 2% SeaKem LE Plus Agarose $ Ethidium bromide (0.5 μg/ml) TBE 8 fi 1,000 4% NuSieve 3:1 Plus Agarose $132 Latitude HT Precast Gels 200 Well (4 50 wells) Alternate well multichannel pipette compatible, 5 gels/box Ethidium bromide (0.5 μg/ml) TAE 400 fi 10,000 1% SeaKem LE Plus Agarose $ Ethidium bromide (0.5 μg/ml) TBE 300 fi 8,000 1% SeaKem LE Plus Agarose $ Ethidium bromide (0.5 μg/ml) TBE 8 fi 1,000 4% NuSieve 3:1 Plus Agarose $ Ethidium bromide (0.5 μg/ml) TBE 100 fi 2,000 2% SeaKem LE Plus Agarose $132 Visit or contact Scientific Support to inquire about custom precast gels. Resolution of DNA markers in a Latitude HT Precast Agarose Gel Alternate loads of bp Marker and 100 bp Ladder (Lonza) run in a 2% SeaKem LE Plus Agarose Gel in 1X TBE buffer containing 0.5 μg/ml ethidium bromide. Gels run at 6 V/cm, 1 hour run using the TruBand Anchor. 28

37 Latitude HT Gels Continued Applications High-throughput DNA analysis Restriction digests PCR Multiplex PCR RT-PCR Genotyping Fingerprinting Quality control for sequencing or microarray construction Library construction Performance and Quality Tests DNase: No activity detected Gel performance: Sharp bands and low background fluorescence Specifications Gels per box: 5 Gel dimensions: 24 cm 14 cm Gel thickness: 6.5 mm Ethidium Bromide: 0.5 μg/ml Tray dimensions: 25 cm 15 cm Well volume: 10 μl 12 μl for 50 well gels 25 μl 30 μl for 25 well gels Chamber Compatibility Information Latitude HT Gels fit most large submerged electrophoresis systems. We recommend the following systems: Owl Centipede Horizontal System Owl Millipede Horizontal System FisherBiotech Wide Format System FB-SB-2318 Bio-Rad Sub-Cell Models 96 and 192 Shelton JSB-96 Storage Conditions 18ºC 26ºC for 6 12 months depending upon agarose concentration Ordering Information Latitude HT Precast Agarose Gels Supporting Products Cat. No. Description Price TruBand Gel Anchors Free with your first order of Latitude Gels Owl Millipede, Shelton JSB-96, $39 Fisher SB Other standard chambers $39 DNA Loading Buffer (6X) (contains bromophenol blue and xylene cyanol) ml $30 AccuGENE 50X TAE Buffer L $67 AccuGENE 10X TAE Buffer L $ L $87 AccuGENE 10X TBE Buffer L $ L $ L $ L $234 AccuGENE 5X TBE Buffer L $ L $ L $152 Precast Gels Application Support Loading and Running DNA in Agarose Gels, Section III, page 89 Detection and Sizing of DNA in Agarose Gels, Section IV, page 95 Related Products DNA Ladders and Markers 36 29

38 Latitude Midigels Versatile medium sized precast gels Precast Gels Latitude Precast Agarose Midigels are designed for high sample throughput DNA analysis applications requiring increased resolution distance. These gels are precision manufactured for rapid and reproducible resolution of DNA sizes from 8 bp to 10 kb. Latitude Gels are available in a variety of well formats and agarose concentrations, in TAE and TBE Buffer. Manufactured with high quality SeaKem or NuSieve Agarose for reliability Latitude Gels fit most midigel chambers and provide optimal performance in the Latitude Chamber 10 cm 15 cm 15 cm Ordering Information Performance of the 40 well Latitude Precast Agarose Midigels Alternate loads of 100 bp DNA Ladder and Lonza 20 bp DNA Ladder (Lonza)(1 μl marker/lane) run in a 4% NuSieve 3:1 Plus Agarose Gel in 1X TBE Buffer containing 0.5 μg/ml Ethidium Bromide. 6 V/cm, 70 minute run in a 10 cm 15 cm Latitude Gel Chamber using the TruBand Gel Anchor. For optimal performance, use Latitude Gels with the TruBand Anchor, see below. Cat. No. Stain Buffer Separation Range (bp) Agarose Price Latitude Midigel 20 Well 8 gels/box Ethidium bromide (0.5 μg/ml) TAE 400 > 10,000 1% SeaKem LE Plus Agarose $ Ethidium bromide (0.5 μg/ml) TBE 300 8,000 1% SeaKem LE Plus Agarose $132 Latitude Midigel 40 Well 8 gels/box Ethidium bromide (0.5 μg/ml) TAE 400 > 10,000 1% SeaKem LE Plus Agarose $ Ethidium bromide (0.5 μg/ml) TAE 100 3,000 2% SeaKem LE Plus Agarose $ Ethidium bromide (0.5 μg/ml) TBE 300 8,000 1% SeaKem LE Plus Agarose $ Ethidium bromide (0.5 μg/ml) TBE 100 2,000 2% SeaKem LE Plus Agarose $ Ethidium bromide (0.5 μg/ml) TBE 8 1,000 4% NuSieve 3:1 Plus Agarose $ cm Visit or contact Scientific Support to inquire about custom precast gels. Performance and Quality Tests DNase: No activity detected Gel performance: Sharp bands and low background fluorescence Storage Conditions 18ºC 26ºC for 6 12 months depending upon agarose concentration Specifications Gels per box: 8 Gel dimensions: 10 cm 15 cm Gel thickness: 6.0 mm Ethidium Bromide: 0.5 μg/ml Tray dimensions: 10.4 cm 15.6 cm Well volume: 10 μl 12 μl Ordering Information Cat. No. Description Price Latitude Midigel Chamber Casting accessories not available Gel Chamber $307 TruBand Gel Anchor Free with your first order of Latitude Gels Standard Chambers $ Latitude Chamber $36 30

39 PAGEr Gold TBE Gels Polyacrylamide minigels for DNA separation PAGEr Gold TBE Precast Gels provide fine resolution of DNA fragments <2,000 bp, and are optimal for resolving 1% differences in DNA fragment size. These ready-to-use gels are specially designed for maximum user convenience,. Opening the cassette requires a simple snap of the comb. Easy-to-load: unique gold colored cassette and marked lanes are easy-to-see Easy-to-open: simple snap-open cassette does not require a special opening device Separation ranges for nucleic acids in PAGEr Gold TBE Gels Polyacrylamide Concentration Size Separation Range 6% 75 bp 2,000 bp 10% 30 bp 1,000 bp 4-20% 10 bp 2,000 bp Ordering Information Description Cassette size 10 well 12 well 16 well PAGEr Gold TBE Gels 10 gels per box $115 6% 9 10 cm cm 10% 9 10 cm cm 4-20% gradient 1,000 bp 700 bp 525 bp 500 bp 400 bp 300 bp 200 bp 9 10 cm cm Performance of PAGEr Gold TBE Gels Precast Gels Applications Fine resolution of PCR products Oligo analysis Performance and Quality Tests Each lot of PAGEr Gold TBE Gels is functionally tested Certificate of Analysis available upon request Specifications Gels per box: 10 Gel dimensions: 8.3 cm 7.1 cm 0.1 cm 8.3 cm 8.3 cm 0.1 cm Cassette thickness: 0.49 cm (9 cm 10 cm tray) 0.55 cm (10 cm 10 cm tray) Cassette dimensions: 9 cm 10 cm (L W) 10 cm 10 cm (L W) Well volume: 10 well 32 μl 12 well 20 μl 16 well 14 μl Storage Conditions 2 C 8 C for 3.5 months from date of manufacture 100 bp 50 bp Alternating lanes of the 50 bp -1,000 bp DNA Marker (2 μl/lane) and 100 bp DNA Ladder (1 μl/lane) (Lonza) separated on a 420% PAGEr Gold TBE Gel. Gel run at 200 V for 60 minutes, stained for 15 minutes in 0.5 μg/ml EtBr, and destained for 5 minutes. See page 72 for chamber compatibility. Application Support Separation of DNA in Polyacrylamide Gels, Section VII, page 125 Related Products AccuGENE Buffers 41 DNA Ladders and Markers 36 DNA Loading Buffer 41 GelStar Nucleic Acid Gel Stain 38 PAGEr Minigel Chamber 50 31

40 Precast Gels and Related Products for RNA Analysis Clean, reliable, guaranteed RNase free Reliant and Latitude Precast RNA Gels Reliant and Latitude Precast RNA Agarose Gels are precision cast in 1.25% SeaKem Gold Agarose with MOPS buffer and are guaranteed RNase-free. These gels are ideal for Northern blotting and analysis of RNA transcripts. Our RNA markers, stains, and buffers are designed to optimize RNA separations. Ordering Information Cat. No. Buffer Description No. of Wells Price Precast Gels 8 Wells Portrait 6 cm 9.5 cm Reliant RNA Gel System 20 minigels/box MOPS 1.25% SeaKem Gold for RNA 8 well $ MOPS 1.25% SeaKem Gold for RNA 20 well (landscape) $144 Cat. No. Gel Description Sample Buffer Description Price Reliant RNA Analysis Kits Contains: Gels and sample buffer described, RNA Marker, 1 L 10X MOPS Buffer, and μl GelStar Stain well Reliant RNA Gel Formaldehyde Sample Buffer $ Wells Landscape 6 cm well Reliant RNA Gel Formaldehyde Sample Buffer $ well Reliant RNA Gel Glyoxal Sample Buffer $ well Reliant RNA Gel Glyoxal Sample Buffer $ cm 20 Wells Portrait or 40 Wells (2 20) Portrait 15 cm Cat. No. Buffer Description No. of Wells Price Latitude RNA Midigels 8 midigels/box MOPS 1.25% SeaKem Gold 2 20 well $ MOPS 1.25% SeaKem Gold 1 20 well $156 NOTE: Due to varying storage requirements, kit components may arrive in separate shipping containers. 10 cm Applications Northern blotting RNA electrophoresis RNA integrity checks Performance and Quality Tests Agarose: No RNase activity detected Gel performance: Sharp RNA bands and low background fluorescence are visible with ethidium bromide, SYBR Green II and GelStar Nucleic Acid Gel Stains Application Support Separation of RNA in Agarose Gels, Section VIII, page 131 Resolution of RNA Markers run in a Reliant RNA Gel 9 kb 6 kb 5 kb 4 kb 3 kb 2.5 kb 2 kb 1.5 kb 1 kb 0.5 kb Gel loaded with samples of RNA Marker 0.5 kb - 9 kb. Marker loaded at 200 ng (lanes 3 and 6) and 1 μg (lanes 1, 4, 5 and 8). Gel run at 5 V/cm for 2 hours using AccuGENE MOPS Buffer (1X). RNA stained for 30 minutes using GelStar Nucleic Acid Gel Stain (1:10,000 dilution). 32

41 Precast Gels and Related Products for RNA Analysis Continued Sample Buffers Ordering Information These ready-to-use buffers are used for denaturation of RNA samples for electrophoresis on Reliant and Latitude Precast RNA Gels, and are ideal for Northern blotting. Storage Conditions Glyoxal: 18 C 26 C for 12 months; storage at 4 C will extend the stability to 2 years Formaldehyde: -20 C for 12 months AccuGENE 10X MOPS Buffer AccuGENE 10X MOPS Buffer is specially formulated for use with Latitude and Reliant Precast Gels for RNA electro phoresis. It is manufactured with the same reagents used in our precast gels. The buffer contains 0.2 M MOPS (free acid), 0.05 M sodium acetate, 0.01 M EDTA (disodium salt), and 0.01 M EGTA (free acid), ph 7.0. Storage Conditions 18 C 24 C Cat. No. Description Price Glyoxal Sample Buffer Ideal for Northern blotting ml $43 Formaldehyde Sample Buffer Ideal for visualization /analysis contains bromophenol blue and xylene cyanol ml $39 Ordering Information Cat. No. Description Price AccuGENE 10X MOPS Buffer L $60 Precast Gels RNA Marker kb RNA Markers kb, are suitable for sizing single stranded RNA in glyoxal or formaldehyde denaturing systems. The RNA Marker consists of ten RNA transcripts: 0.5, 1, 1.5, 2, 2.5, 3, 4, 5, 6, and 9 kb in length. Markers can be denatured with standard procedures, and visualized on Northern blots with labeled lambda sequence. Detect: 4 μg with ethidium bromide, or smaller quantities with GelStar or SYBR Green II Gel Stains. Storage Conditions -80 C for 24 months or -20 C for 6 months Ordering Information Cat. No. Description Price RNA Marker kb μg (1 μg/μl) $133 Resolution of RNA Markers run in a Reliant RNA Gel 9 kb 6 kb 5 kb 4 kb 3 kb 2.5 kb 2 kb 1.5 kb 1 kb 0.5 kb Related Products FlashGel System for RNA 22 33

42 Precast Gels and Related Products for RNA Analysis Continued GelStar Nucleic Acid Gel Stain GelStar Nucleic Acid Gel Stain is a fast-acting, fluorescent stain that is up to fifteen times more sensitive than Ethidium Bromide for RNA detection. Detects 3 ng of RNA or 20 pg of dsdna Ordering Information Cat. No. Description Price GelStar Nucleic Acid Gel Stain μl $146 Product licensed from Molecular Probes, Inc. Precast Gels RNA Detection with GelStar Stain A B C Samples of E. coli total RNA were denatured using the following denaturants: Lane A: Formaldehyde/ Formamide; Lane B: Formamide; Lane C: Glyoxal. Samples were loaded at 2 μg/lane for the formaldehyde/formamide and formamide only denatured samples, and 4 μg/lane for the glyoxal denatured samples. Reliant RNA Precast Agarose Gels were run at 7 V/cm for 40 minutes in 1X MOPS Buffer and post stained with GelStar Gel Stain and photographed on the Clare Chemical Research, Inc., Dark Reader Transilluminator. GelStar Gel Stain Photographic Filter inch square $58 (Wratten #9) SYBR Green II Nucleic Acid Gel Stain SYBR Green II Nucleic Acid Gel Stain is a highly sensitive fluorescent stain that is ideal for detection of RNA. Detects 2 ng of RNA or 100 pg of dsdna Storage Conditions -20 C for stain 18 C 26 C for photographic filter RNA Detection with SYBR Green II Stain A B C Ordering Information Cat. No. Description Price SYBR Green II Nucleic Acid Gel Stain μl $ μl $379 Product licensed from Molecular Probes, Inc. SYBR Green Gel Stain Photographic Filter inch square $58 (Wratten #15) Samples of E. coli total RNA were denatured using the following denaturants: Lane A: Formaldehyde/ Formamide; Lane B: Formamide; Lane C: Glyoxal. Samples were loaded at 2 μg/lane for the formaldehyde/formamide and formamide only denatured samples, and 4 μg/lane for the glyoxal denatured samples. Reliant RNA Precast Agarose Gels were run at 7 V/cm for 40 minutes in 1X MOPS Buffer and post stained with SYBR Green II Gel Stain and photographed on the Clare Chemical Research, Inc., Dark Reader Transilluminator. 34

43 Markers, Stains and Buffers Optimal performance and convenience Great performance starts with high quality agarose and gels, but for complete assurance, you need to use high quality markers, ladders, stains, and buffers. Lonza supports a broad offering of products that complement and match the performance of our agarose and precast gels. Rapidly estimating fragment size requires clear sharp banding patterns on each and every gel. We offer two types of ladders and markers: Standard and SimplyLoad. Standard Markers and Ladders are ready to dilute prior to loading your gel, while our convenient SimplyLoad Ladders are premixed, ready for direct loading. Our DNA quantitation ladders are ideal for the accurate estimation of molecular mass of fragments from 10 ng to 100 ng. Seeing all of your data is critical to the overall success of your experiment. GelStar Nucleic Acid Gel Stain clearly detects fragments down to 20 pg of DNA. Maximize your performance by adding the stain directly to your gel prior to casting or post-stain your gel. We also offer SYBR Green Nucleic Acid Gel Stains. Finally, Lonza offers a complete line of AccuGENE Electrophoresis and Molecular Biology Buffers to support your research. Our AccuGENE Buffers are formulated to optimize performance of our agarose and precast gel products. Markers, Stains and Buffers 35

44 DNA Ladders and Markers Sizing made easy 500 bp 200 bp 1,000 bp 500 bp 1,000 bp 700 bp 525 bp 500 bp 400 bp 300 bp 2,500 bp 1,500 bp 1,000 bp 700 bp 525 bp 500 bp 400 bp 1,000 bp 500 bp 3,000 bp 200 bp 200 bp 300 bp 1,000 bp 100 bp 200 bp 100 bp 100 bp 100 bp 100 bp 500 bp 50 bp 50 bp 20 bp 20 bp 100 bp Markers, Stains and Buffers Cat. No. Standard SimplyLoad Ladders 20 bp bp Ext ,000 bp N/A 50-2,500 bp N/A 100 bp bp Ext ,000 bp 5,000 bp 1,000 bp 100 ng 700 bp 70 ng 1,000 bp 10 ng 700 bp 20 ng 9 kb 3,000 bp 10 kb 500 bp 50 ng 500 bp 50 ng 6 kb 5 kb 2,000 bp 7 kb 5 kb 4 kb 4 kb 3 kb 3 kb 2.5 kb 1,000 bp 1,000 bp 2.5 kb 2 kb 200 bp 20 ng 200 bp 70 ng 2 kb 1.5 kb 1.5 kb 100 bp 10 ng 100 bp 100 ng 1 kb 1 kb 0.5 kb 500 bp 500 bp Cat. No. Standard SimplyLoad Ladders Tandem N/A bp kb N/A QuantLadder Reverse QuantLadder RNA Ladder N/A 36 SimplyLoad Ladders are supplied in ready-to-load concentrations. See page 96 for detailed size information

45 DNA Ladders and Markers Continued Standard Ladders and Markers are ready-to-dilute prior to loading on your gel. Plasmid-free to ensure minimal background. Ordering Information Standard DNA Ladders SimplyLoad Ladders are supplied ready-to-load on your gel. No need for mixing, heating or diluting prior to loading. Plasmid-free to ensure minimal background. Ordering Information SimplyLoad DNA Ladders Ready-to-dilute, Evenly spaced bands, 200 ng/μl concentration unless otherwise noted Cat. No. Volume Description Applications Price 20 bp DNA Ladder μl 20 bp 500 bp 150 $ bp Extended Range DNA Ladder μl 20 bp 1,000 bp 100 $ bp DNA Ladder Concentration: 100 ng/μl μl 100 bp 1,000 bp 100 $ bp Extended Range DNA Ladder μl 100 bp 3,000 bp 100 $ bp DNA Ladder μl 500 bp 8,000 bp 200 $102 Standard Quantitation Ladders Ideal for estimating molecular mass, 200 ng/μl concentration Ready-to-use, Evenly spaced bands, 200 ng/μl concentration Cat. No. Volume Description Applications Price SimplyLoad 20 bp DNA Ladder μl 20 bp 500 bp 100 $109 SimplyLoad 20 bp Extended Range DNA Ladder μl 20 bp 1,000 bp 100 $109 SimplyLoad 100 bp DNA Ladder μl 100 bp 1,000 bp 100 $109 SimplyLoad 100 bp Extended Range DNA Ladder μl 100 bp 3,000 bp 100 $109 SimplyLoad 500 bp DNA Ladder μl 500 bp 8,000 bp 100 $109 SimplyLoad Tandem DNA Ladder μl 100 bp 12,000 bp 100 $109 Markers, Stains and Buffers Cat. No. Volume Description Applications Price DNA QuantLadder μl 100 bp 1,000 bp 50 $103 DNA Reverse QuantLadder μl 100 bp 1,000 bp 50 $103 Standard DNA Markers Ready-to-dilute, 90 ng/μl concentration Cat. No. Volume Description Applications Price DNA Marker 50 1,000 bp μl 50 bp 1,000 bp 50 $149 DNA Marker 1 10 kb μl 1 kb 10 kb 100 $173 DNA Marker 50 2,500 bp μl 50 bp 2,500 bp 50 $173 SimplyLoad Quantitation Ladders Ideal for estimating molecular mass, 200 ng/μl concentration Cat. No. Volume Description Applications Price SimplyLoad DNA QuantLadder μl 100 bp 1,000 bp 100 $109 SimplyLoad DNA Reverse QuantLadder μl 100 bp 1,000 bp 100 $109 Storage Conditions Standard Ladders and Markers: 4 C or -20 C SimplyLoad Ladders: 4 C Related Products Agarose 4 Precast Gels 17 37

46 GelStar Nucleic Acid Gel Stain Exquisitely sensitive in-gel stain for DNA and RNA Markers, Stains and Buffers GelStar Nucleic Acid Gel Stain is a highly sensitive fluores cent stain for detecting both DNA and RNA. Add GelStar Stain to your agarose solution prior to casting, or post-stain your gels. GelStar Stain exhibits exceptional signal-to-noise ratio with minimal background. Maximum sensitivity, detect as little as 20 pg of dsdna or 3 ng of RNA Versatile, use for agarose or polyacrylamide gel electrophoresis, ideal alternative to silver staining Ultimate user flexibility, add GelStar Stain prior to gel casting or post-stain, no destaining required Complete staining solution for all types of nucleic acids Detect fragments with either a standard 300 nm UV transilluminator or the Clare Chemical Research, Inc., Dark Reader Transilluminator Document your results with either Polaroid or CCD-based Camera Systems GelStar Stain versus Ethidium Bromide GelStar Stain Ordering Information Cat. No. Size Price GelStar Nucleic Acid Gel Stain μl $146 Supplied as a 10,000X concentrated solution in DMSO. Product licensed from Molecular Probes, Inc. Cat. No. Description Price GelStar Gel Stain Photographic Filter inch square $58 (Wratten #9) GelStar Gel Stain Photographic Filter Use for optimal sensitivity with black and white film Suitable for use with most Polaroid Documentation or Camera Systems Applications DNA and RNA detection SSCP and heteroduplex analysis Storage Conditions -20 C for stain 18 C 26 C for photographic filter Ethidium Bromide Application Support Detecting DNA with Gelstar and SYBR Green Nucleic Acid Stains. Section IV, pages 98, Detecting DNA in Polyacrylamide Gels with Gelstar and SYBR Green Nucleic Acid Stains, Section VII, page 127 Detection of RNA in Agarose Gels, Section VIII, page Serial dilution of SimplyLoad DNA QuantLadder on 2% Reliant Precast Gels post-stained with 1X GelStar Stain (left) or 0.5 g/ml Ethidium Bromide (right) for 45 minutes. Related Products AccuGENE Buffers 41 Agarose 4 DNA Ladders 36 MDE Gel Solution 44 Precast Gels 17

47 SYBR Green Nucleic Acid Gel Stains Sensitive fluorescent stains for DNA and RNA SYBR Green Nucleic Acid Gel Stains are fluorescent stains for detecting DNA and RNA, exhibiting excellent signal-to-noise ratio with minimal background. SYBR Green Stains are more sensitive than standard stains, making them convenient alternatives to silver staining and radioisotopes. For maximum detection, gels should be post-stained and photographed with the SYBR Green Photographic Filter. SYBR Green I Stain Detects as little as 60 pg of dsdna and 1 ng oligonucleotides Optimal for PCR, apoptosis studies, and heteroduplex analysis SYBR Green II Stain Detects 100 pg of ssdna and 2 ng of RNA Optimal for RNA gel electrophoresis and SSCP analysis Ordering Information Cat. No. Size Price SYBR Green I Nucleic Acid Gel Stain μl $ μl $379 Supplied as a 10,000X concentrated solution in DMSO. Product licensed from Molecular Probes, Inc. SYBR Green II Nucleic Acid Gel Stain μl $ μl $379 Supplied as a 10,000X concentrated solution in DMSO. Product licensed from Molecular Probes, Inc. SYBR Green Gel Stain Photographic Filter inch square $58 (Wratten #15) SYBR Green Gel Stain Photographic Filter Required for optimal sensitivity with black and white film Suitable for use with most Polaroid Systems RNA Detection with SYBR Green II Stain A B C DNA stained with SYBR Green I Stain or ethidium bromide SYBR Green I Stain Ethidium Bromide Stain ng DNA Markers, Stains and Buffers Samples of E. coli total RNA were denatured using the following denaturants: Lane A: Formaldehyde/ Formamide; Lane B: Formamide; Lane C: Glyoxal. Samples were loaded at 2 μg/lane for the formaldehyde/formamide and formamide only denatured samples, and 4 μg/lane for the glyoxal denatured samples. Reliant RNA Precast Agarose Gels were run at 7 V/cm for 40 minutes in 1X MOPS Buffer and post stained with SYBR Green II Gel Stain and photographed on the Clare Chemical Research, Inc., Dark Reader Transilluminator. Applications DNA and RNA detection SSCP and heteroduplex analysis Storage Conditions -20 C for stain 18 C 26 C for photographic filter DNA samples (pbr322 Msp I digest) ranging from 1 to 200 ng per lane were separated on a 10 cm 16 cm 0.1 cm, 4% vertical MetaPhor Agarose gel prepared in 1X TBE Buffer. The gel was run for 1 hour at 488 V/cm. Following electrophoresis the gel was divided into two, and one half was stained with 1 μg/ml ethidium bromide while the other was stained with SYBR Green I Stain (1:10,000 dilution of stock). Detection was achieved with standard 300 nm UV transillumination. Related Products AccuGENE Buffers 41 Agarose 4 MDE Gel Solution 44 Precast Gels 17 39

48 AccuGENE Molecular Biology Buffers Convenient and ready-to-use Markers, Stains and Buffers AccuGENE Molecular Biology Buffers are ready-to-use solutions ideal for a wide range of molecular biology applications. Reliable Manufactured according to strict quality control standards to ensure lot-to-lot consistency High quality Guaranteed DNase, RNase, and protease-free Efficient Ready-made solutions eliminate experiment preparation time Flexible Customized solutions are available to meet individual needs Storage Conditions 18 C 24 C Ordering Information Cat. No. Size Price AccuGENE Molecular Biology Water L $ L $ L $ L $204 AccuGENE 0.5 M EDTA Solution Disodium salt, ph ml $ L $62 AccuGENE 5 M Sodium Chloride L $28 Ordering Information Cat. No. Size Price AccuGENE 1X TE Buffer 0.01 M Tris, M EDTA (disodium salt), ph ml $ L $149 AccuGENE 1 M Tris HCl Buffer L $ L $ L $34 AccuGENE LB Broth (Luria Bertani Medium) 10 g/l Bacto-Tryptone, 5 g/l Bacto-Yeast Extract, and 10 g/l NaCl ml $23 AccuGENE Super Broth (Terrific Broth) 12 g/l Bacto-Tryptone, 24 g/l Bacto-Yeast Extract, 6.3 g/l glycerol, 2.5 g/l K 2 HPO 4, and 3.8 g/l KH 2 PO 4, ph ml $ L $51 AccuGENE Neutralization Solution 1.5 M NaCl, 1.0 M Tris, ph L $ L $206 AccuGENE 1X PBS 1.7 mm KH 2 PO 4, 5 mm Na 2 HPO 4, 150 mm NaCl, ph L $30 AccuGENE 10X PBS M KH 2 PO 4, 0.05 M Na 2 HPO 4, 1.5 M NaCl, ph 7.4 AccuGENE 10% SDS Monosodium salt ml $ ml $38 AccuGENE 3 M Sodium Acetate, ph ml $45 AccuGENE 20X SSC Buffer 3.0 M NaCl, 0.3 M sodium citrate, ph L $ L $ L $287 AccuGENE 20X SSPE Buffer 3.0 M NaCl, 0.2 M NaH 2 PO 4 H 2 O, 0.02 M EDTA, ph L $ L $ L $246

49 AccuGENE Electrophoresis Buffers Optimal performance AccuGENE Electrophoresis Buffers are formulated for maximum performance and convenience, and are optimized for use with our agarose and precast gels. AccuGENE Buffers for DNA, RNA, and protein electrophoresis are prepared with high quality reagents and use 18 megohm water. Products are filtered using a 0.2-micron filter, and are guaranteed DNase/RNase free. Reliable Manufactured according to strict quality control standards to ensure lot-to-lot consistency Efficient Ready-to-use solutions eliminate experiment preparation time Flexible Customized solutions are available to meet individual needs Storage Conditions 18 C 24 C 4 C for CE Buffer Ordering Information Buffers for Capillary DNA Sequencing Cat. No. Size Price AccuGENE 10X CE Buffer Formulated for use on the ABI Prism 3700 DNA Analyzer L $856 AccuGENE 10X CE Buffer Formulated for use on the ABI Prism 3100 Genetic Analyzer ml $94 Buffers for Protein Electrophoresis Cat. No. Size Price AccuGENE 10X Tris-Glycine Buffer 0.25 M Tris base, 1.92 M Glycine L $ L $81 Application Support Buffers for Electrophoresis, Section II, page 81 Ordering Information Buffers for DNA Electrophoresis Cat. No. Size Price AccuGENE 10X TAE Buffer 0.4 M Tris-acetate, 0.01 M EDTA (disodium salt), ph L $ L $87 AccuGENE 10X Tris-Glycine SDS Buffer 0.25 M Tris base, 1.92 M Glycine, 1% SDS L $ L $81 Buffers for RNA Electrophoresis Cat. No. Size Price AccuGENE 10X MOPS Buffer 0.2 M MOPS (free acid), 0.05 M sodium acetate, 0.01 M EDTA (disodium salt), 0.01 M EGTA (free acid), ph 7.0. No detectable RNase activity L $60 Electrophoresis Loading Buffers Markers, Stains and Buffers AccuGENE 50X TAE Buffer 2.0 M Tris-acetate, 0.05 M EDTA, ph L $67 AccuGENE 5X TBE Buffer 0.45 M Tris-borate, 0.01 M EDTA (disodium salt), ph L $ L $ L $152 Cat. No. Size Price DNA Loading Buffer (6X) Ficoll based with bromophenol blue and xylene cyanol ml $30 Triple-Dye Loading Buffer (6X) Contains bromophenol blue, xylene cyanol, and orange G ml $35 AccuGENE 10X TBE Buffer 0.89 M Tris-borate, 0.02 M EDTA (disodium salt), ph L $ L $56 BE08-238Z4 4 L Europe only BE08-238Z10 10 L Europe only L $ L $234 Related Products Agarose 4 Long Ranger Gel Solutions 43 MDE Gel Solution 44 Precast Gels 17 Protein Electrophoresis Products 45 41

50 GelBond Film Agarose support film GelBond Film is a transparent, flexible polyester film designed to support agarose gels. Gels cast on GelBond Film remain permanently attached to the film through electrophoresis or immunodiffusion and all subsequent fixing, staining, destaining, and drying procedures (gels remain flexible after drying). GelBond Film is available either as precut sheets or rolls. Reliable Agarose gels cast on GelBond Film retain their original dimensions during staining and after drying Durable Gels, particularly thin ones, are easier to handle during staining, destaining, and drying when supported Convenient Gel orientation can be recorded directly on the GelBond Film prior to casting NOTE: Polyester films will not transmit light of less than 310 nm, and will fluoresce at higher wavelengths. Applications Drying and support of agarose gels Storage Conditions 18 C 26 C GelBond PAG Film Ordering Information Cat. No. Sheet Size (mm) Chamber Compatibility Price GelBond Film Sheets for agarose gels; 0.2 mm thick sheets, 100 per package $ $ Bio-Rad Wide Mini-Sub Cell, $289 Sub-Cell (H) $ Hoefer SE400, SE600 (V) $514 Bio-Rad PROTEAN II xi (V) $ GE Multiphor (H) $553 Custom-cut GelBond Film is available upon special request. Please inquire for pricing and availability. (H) = Horizontal; (V) = Vertical Cat. No. Roll Width Price GelBond Film Rolls for agarose gels; 0.2 mm thick rolls, 16.5 meters long mm $ mm $ mm $ mm $579 Related Products Agarose 5 Gel Support Films 42 Polyacrylamide support film GelBond PAG Film is a transparent, flexible polyester film designed to support polyacrylamide or MDE Gels. The acrylamide monomers covalently attach to the coating on the film during the polymerization reaction. Gels remain permanently attached to the film through electrophoresis and all subsequent fixing, staining, destaining, and drying procedures. Reliable Polyacrylamide gels retain their original dimensions during staining and after drying Durable Gels, particularly thin ones, are easier to handle during staining, destaining, and drying when supported Convenient Gel orientation can be recorded directly on the GelBond PAG Film prior to casting NOTE: Polyester films will not transmit light of less than 310 nm, and will fluoresce at higher wavelengths. Applications Drying and support of polyacrylamide gels Storage Conditions 18 C 26 C, protect from light Ordering Information Related Products MDE Gel Solution 44 ProSieve 50 Acrylamide Gel Solution 60 Cat. No. Sheet Size (mm) Chamber Compatibility Quantity (sheets) GelBond PAG Film Sheets GelBond PAG Film sheets for polyacrylamide gels; 0.2 mm thick sheets Price $ Hoefer SE400, SE600, 50 $289 Bio-Rad PROTEAN II GE Multiphor 50 $ $ $ GE Multiphor II 50 $ Biometra SA $ X-ray size 10 $289 Custom-cut GelBond PAG Film is available on special request. Please inquire for pricing and availability.

51 Long Ranger Sequencing Products Reliable and convenient slab gel sequencing Long Ranger products are designed for high performance sequencing on ABI Prism 377 and manual sequencers. Long Ranger Gel Solution offers exceptional results for sequencing and STR analysis Singel Packs contain all necessary reagents for casting a single gel in a pre-measured, ready-to-use pouch Gel Slick Solution is a safe alternative to hazardous silane-based coating solutions and is suitable for sequencing gels and coating microcentrifuge tubes Applications DNA and RNA sequencing STR analysis Identity testing Genotyping Differential display Storage Conditions 18 C 26 C Ordering Information Cat. No. Size Price Long Ranger 50% Gel Solution (50% concentrate) ml $ L $820 Gel Slick Solution ml $102 Cat. No. Quantity/box Price Long Ranger Singel Pack for ABI Sequencers cm $132 Long Ranger Singel Pack for ABI Sequencers cm $132 Long Ranger Singel Pack for LI-COR Sequencers 33 or 41 cm $132 Long Ranger Singel Pack for manual sequencing Comparison of Long Ranger Gel Solution and polyacrylamide on the ABI Prism 377 Automated DNA Sequencer Long Ranger Gel Solution Polyacrylamide $132 Singels instrument compatibility Cat. No. Instrument Plate Length Gel Matrix ABI Prism % Long Ranger Singel Pack, 6 M urea % Long Ranger Singel Pack, 6 M urea LI-COR 33 or 41 6% Long Ranger Singel Pack, 7 M urea Manual Sequencing See page 41 for sequencing buffers. 5.75% Long Ranger Singel Pack, 7 M urea Sequencing and Mutation Detection Products This comparison used 5% Long Ranger Gel Solution, 4% polyacrylamide, 36 cm well-to-read, Amplitaq DNA Polymerase, FS, dye primer on M13 mp18 template. Typical Results on the ABI Prism DNA 377 Automated DNA Sequencer Long Ranger Gel Solution % Gel WTR Run Condition Read 98.5% 5% 36 cm 3 kv 3.5 hrs 600 5% 36 cm 1.68 kv 9 hrs 780 5% 48 cm 2.88 kv7.5 hrs 820 Related Products AccuGENE Buffers 41 43

52 MDE Heteroduplex Kit An ideal solution for heteroduplex analysis Mutation Detection Enhancement (MDE ) Gel Solution is a highly sensitive, high resolution, unique gel formulation designed to separate based on conformational differences. Increased sensitivity means you will find more mutations than standard polyacrylamide gels. Detects more mutations. Unique formulation Easy-to-cast and handle, especially with GelBond PAG Film Eliminate messy silver staining by using GelStar Nucleic Acid Gel Stain Greater flexibility; can be used on standard manual or automated equipment Try our complete MDE Heteroduplex Kit containing MDE Gel Solution, Heteroduplex Control DNA, Triple Dye Loading Buffer, GelStar Nucleic Acid Gel Stain, and AccuGENE 10X TBE Buffer NOTE: Protocols are available for both manual and automated hetero duplex and SSCP analysis; contact Scientific Support or visit research for more information. Performance of a 1.0X MDE Gel for heteroduplex analysis Ordering Information Cat. No. Size Price MDE Mutation Detection Enhancement Heteroduplex Kit Kit $514 MDE Mutation Detection Enhancement Gel Solution ml $264 Heteroduplex Control DNA μl $151 Triple Dye Loading Buffer (6X) contains bromophenol blue, xylene cyanol, and orange G ml $35 GelStar Nucleic Acid Gel Stain μl $146 Product licensed from Molecular Probes, Inc. GelStar Gel Stain Photographic Filter inch square $58 (Wratten #9) AccuGENE 10X TBE Buffer Sequencing and Mutation Detection Products CFTR exon 10 mutations were analyzed in a 1X MDE Gel (20 cm 40 cm 1 mm) run in 0.6X TBE Buffer at 800 volts for 3 hours. The voltage was increased to 1,000 volts for 18 hours (20,400 V/hr total). The gel was silver stained following a standard protocol. Lane 1: 100 bp ladder Lane 2: M470V 1,1 (homozygote) Lane 3: M470V 2,2 (homozygote) Lane 4: M470V 1,2 (heterozygote) Lane 5: F508/ F508 (3 base deletion) Lane 6: F508/WT Lane 7: I507 (3 base deletion) / WT From Clinical Chemistry 39: , Lane 8: F508/I506V (1 base substitution) Lane 9: F508/F508C (1 base substitution) Lane 10: F508/Q493X (1 base substitution) Lane 11: I506V/ WT Lane 12: F508C/ WT Lane 13: 100 bp ladder L $20 AccuGENE SSCP Stop Solution ml $43 Applications Heteroduplex analysis SSCP analysis ( 300 bp) Storage Conditions MDE Gel Solution: 18 C 26 C MDE Heteroduplex Kit: See components Heteroduplex Control DNA: 2 C 8 C Triple Dye Loading Buffer: 2 C 8 C GelStar Nucleic Acid Gel Stain: -20 C AccuGENE 10X TBE Buffer: 18 C 26 C GelStar Photographic Filter: 18 C 26 C Related Products Gel Slick Solution 43 GelBond PAG Film 42 44

53 Protein Electrophoresis Chapter 2

54 Protein Analysis Protein Electrophoresis Protein Gel Electrophoresis Introduction 47 PAGEr Precast Gels for Protein Electrophoresis 48 PAGEr Minigel Chamber 50 Protein Markers, Buffers, and Stains 51 IsoGel Agarose and Precast Gels for Isoelectric Focusing 56 Agarose for Protein Separation 58 ProSieve 50 Acrylamide Gel Solution 60 GelBond PAG Support Film 61 46

55 Protein Gel Electrophoresis High performance products that are fast and easy to use Protein Electrophoresis Lonza offers a versatile line of high performance protein electrophoresis products. Our precast gels, buffers, markers and stains are optimized to work together to provide the sharpest resolution and the greatest sensitivity, in the fastest, most convenient formats. Ask about our custom capabilities to create the precise format for your research requirements Fresh Gels Every Time PAGEr Precast Gels ProSieve Markers AccuGENE Buffers SYPRO Stains 47

56 PAGEr Precast Gels for Protein Electrophoresis PAGEr Precast Gels for Protein Electrophoresis Reliable, easy-to-use minigels PAGEr Precast Gels are easy-to-use protein minigels that offer sharper resolution, more consistent protein transfer, and longer shelf life than any other Tris-Glycine gel. PAGEr Gels are easy-to-use and compatible with most minigel chambers. Applications Western blotting Denaturing and native protein electrophoresis 2D electrophoresis Performance and Quality Tests Every lot of PAGEr Precast Gels is functionally tested and 100% guaranteed PAGEr Precast Gel comb formats Comb configurations are designed for a range of sample volumes and throughput, including multichannel pipette compatible formats. Lonza offers over 70 format options for denatured and native protein separation over a wide molecular weight range, in an array of configurations in both 9 cm 10 cm and 10 cm 10 cm sizes to fit popular chambers. See chamber compatibility chart (below) to determine the right gel size for your system. Razor sharp resolution Crisp separation of proteins 5 kda 250 kda Easy to use Marked sample lanes for easy loading and simple twist open design Compatible 2 sizes to fit most chambers Versatile Multiple well formats and gel concentrations Tris-Glycine buffer Traditional Laemmli separation Fresh We ship fresh gels every time for guaranteed performance Fresh Gels Every Time Number of wells: Recommended load volu Number of wells: Recommended load volu Number of wells: Recommended load volume: 550 μl sample, or 30 μl sample 7 cm IPG strip, 12 μl marker 12 μl marker *Multichannel pipette compatible Specifications Cassette Dimensions Cassette Thickness Gel Dimensions 9 cm 10 cm (L W) 0.49 cm (L W) 0.1 cm 10 cm 10 cm (L W) 0.55 cm (L W) 0.1 cm Gel Matrix/Buffer: Polyacrylamide/Tris-Glycine Note: Gels do not contain SDS. Add SDS to sample buffer to create denaturing running conditions. Stacking Gel: 4% stacking gel Well formats: 2D well, 8+1 well*, 10 well, 12 well, 16 well, 17 well* *multichannel pipette compatible well formats Cassettes: Plastic Storage/Shelf Life 2 C 8 C for 3.5 months from date of manufacture Guaranteed 10 weeks shelf life upon receipt Chamber Compatibility PAGEr Precast Gels are available in 9 cm 10 cm and 10 cm 10 cm sizes and fit most standard mini-vertical systems. Some chambers may require modifications for optimal fit with PAGEr Precast Gels. See page 164 or contact Scientific Support for additional information. Standard Vertical Systems PAGEr Gels PAGEr Minigel Chamber 9 cm 10 cm 10 cm 10 cm gels Bio-Rad MiniPROTEAN II, MiniPROTEAN 3 or 9 cm 10 cm gels Ready Gel Cell Systems Reverse the inner core gasket so the flat side faces outward. Novex XCell SureLock Mini-Cell 9 cm 10 cm 10 cm 10 cm gels Request the spacer for the XCell SureLock Mini-Cell Chamber from Scientific Support, (Cat. No ). FisherBiotech Vertical Minigel FBVE121, 10 cm 10 cm gels Owl Separations Systems Wolverine P82 Chamber comes with 2 sets of wedges. Use the thinner wedges for the PAGEr Gold Gels. FisherBiotech Vertical Minigel FB-VE101, 10 cm 10 cm gels Owl Separations Systems Penguin Model P8DS Request adaptor for these chambers from Scientific Support, (Cat. No ). Hoefer Mighty Small (SE250) 9 cm 10 cm 10 cm 10 cm gels Replace the buffer chamber with a Deep lower buffer chamber for the SE260, order number , from GE Healthcare. Daiichi 2, ISS chambers 10 cm 10 cm gels See page 314 for modification instructions. Novex XCell II 9 cm 10 cm or 10 cm 10 cm gels Hoefer Mighty Small (SE260) 9 cm 10 cm or 10 cm 10 cm gels EC 120 Mini Vertical Gel System 9 cm 10 cm or 10 cm 10 cm gels Biometra Mini V Chamber 9 cm 10 cm gels CBS Scientific MGV System, 9 cm 10 cm gels (10 cm 8 cm units) Sigma-Aldrich Mini Techware 10 cm 10 cm gels (11.3 cm 10 cm units) Zaxis System cm 10 cm gels Hoefer Mini VE 10 cm 10 cm gels 48

57 Selecting the Best Precast Gel Ordering Information PAGEr Gold Tris-Glycine Precast Gels 10 gels per box $107 Gel Concentration/ Separation Range 4-12% gradient kda 4-20% gradient kda 8-16% gradient kda 10-20% gradient kda 7.5% kda 10% kda 12% kda 15% kda Cassette size (cm) D well 10 well 12 well 16 well 17 well* well* PAGEr Precast Gels for Protein Electrophoresis PAGEr Gold Scouting Kit Cat. No. Size Quantity Price cm 10 cm Select 6 gels of any type $ cm 10 cm Select 6 gels of any type $70 *For added convenience, our 8+1 and 17-well comb formats can be loaded with a multichannel pipette. Gel concentration and size separation range Lower concentrations are best for resolving large molecules and higher concentrations are best for resolving small molecules. Gradient gels are best for proteins that are unknown or occur over a wide molecular weight range. Percentage PAGEr Precast Gel Gel Concentration Separation Pattern % 12% 10% 7.5% 10-20% 4-20% 8-16% 4-12% Separation Size Range kda kda kda kda kda kda kda kda Gels were run at 175 volts until the dye front reached the bottom of the gel (approx. 60 minutes). 8 μl 10 μl of marker was loaded per lane (0.8 μg 1 μg per band). Gels were stained with Coomassie Brilliant Blue Stain. 49

58 PAGEr Minigel Chamber PAGEr Minigel Chamber Absolute simplicity and optimal performance PAGEr Minigel Chamber The PAGEr Minigel Chamber is the easiest chamber you will ever use. The chamber is designed to provide optimized performance from PAGEr Precast Gels and will also work with most other precast minigels. The simple, lock-in-place core design assures a tight, flat fit and eliminates the risk of buffer leaks. No need to remove the core simply insert gels, close the clamps, fill with buffer and run. Runs one or two gels and accommodates a tank blotting module. Easy-to-use, lock-in-place core eliminates leaking and minimizes handling Perfect fit with 9 cm 10 cm and 10 cm 10 cm PAGEr Gels Even electrical force ensures straight lanes Solid, robust construction Optimizes performance of PAGEr Gels Ordering Information Cat. No. Description Price PAGEr Minigel Chamber PAGEr Minigel Chamber $ PAGEr Blot Module $ PAGEr Minigel Chamber and Blot Module Kit* $662 * Includes chamber, 2 blotting cassettes, and sponge pads (8/pack). Contact Science Support for information about replacement parts. PAGEr Blot Module 50 The PAGEr Blot Module works directly in the PAGEr Minigel Chamber and provides exceptional blotting with a fast, simple protocol. Color-coded cassettes ensure proper orientation of the gel during transfer Transfer time 90 minutes or less Hinged cassette design for easy assembly The system can be purchased as a kit, including the PAGEr Minigel Chamber and PAGEr Blot Module, or components may be purchased separately. Applications SDS-PAGE electrophoresis 2D electrophoresis Tank blotting Specifications Gel types: Gel sizes: Chamber capacity: Buffer volume: Most standard precast minigels (casting apparatus not included) 9 cm 10 cm (adapter included) and 10 cm 10 cm Single gel (blank included), 2 gels, or blot cassettes 800 ml Application Support Tank Blotting PAGEr Precast Gels, Section XI, page 174 Performance of the PAGEr Minigel Chamber kda Markers and E. coli lysate run on a 9 cm 10 cm PAGEr 200 V for 60 minutes in the PAGEr Minigel Chamber. Samples from left to right: 1 & 2 ProSieve Color Protein Marker; 3 8 E. coli lysate; 9 & 10 ProSieve Protein Marker. Related ProductsE AccuGENE Buffers 52 PAGEr Precast Gels 48 ProSieve Protein Markers 51 SYPRO Protein Gel Stains 53

59 ProSieve Color Protein Marker Sharp, accurate confirmation of protein transfer ProSieve Color Protein Markers are ideal for monitoring protein separation prior to staining and provide accurate confirmation of protein transfer in Western blotting. Convenient Just add water and load Sharp Multi-colored, readily identifiable band pattern for monitoring electrophoresis and confirming protein transfer ProSieve Color Protein Markers are a set of proteins and dyes for use as visible markers in SDS-PAGE gels. During electrophoresis, these markers help monitor the e ciency of separation. In Western blotting, they confirm transfer has occurred from the gel to the membrane. The proteins have been labeled with fluorescent dyes and are supplied as lyophilized solids containing the buffer salts and detergent found in the typical Laemmli buffer system. ProSieve Color Protein Markers contain 9 proteins with approximate masses of 10 kda, 15 kda, 20 kda, 25 kda, 40 kda, 50 kda, 80 kda, 125 kda, and 190 kda. Each lot is individually calibrated against unstained ProSieve Protein Markers. Storage Conditions -20 C NOTE: Not recommended for accurate protein sizing. For sharp, accurate sizing, use ProSieve Protein Markers (page 52). ProSieve Color Protein Markers kda Ordering Information Cat. No. Size Price ProSieve Color Protein Marker μl $ μl $123 ProSieve Color Protein Marker performance vs. leading competitors kda Markers were run on a Lonza 4-20% PAGEr Gold Precast Gel in Tris-Glycine SDS Buffer at 125 V for 75 minutes. 1. ProSieve Color Protein Marker 2. ProSieve Color Protein Marker 3. BenchMark Prestained Protein Ladder (Invitrogen) 4. SeeBlue Plus2 Pre-Stained Protein Standard (Invitrogen) 5. Multi-Mark Multi-Colored Standard (Invitrogen) 6. ProSieve Color Protein Marker 7. ProSieve Color Protein Marker 8. Kaleidoscope Prestained Standards (Bio-Rad) 9. Full Range Rainbow Mol. Wt. Markers (Amersham Pharmacia) 10. High Range Color Markers (Sigma) 11. ProSieve Color Protein Marker 12. ProSieve Color Protein Marker Protein Markers, Buffers, and Stains Typical results Related Products AccuGENE Buffers 52 PAGEr Precast Gels 49 ProSieve 50 Acrylamide Gel Solution 60 51

60 ProSieve Protein Marker Protein Markers, Buffers, and Stains Sharp, accurate sizing of proteins 5 kda 225 kda ProSieve Protein Marker consists of a novel set of proteins designed for accurate sizing of protein samples in SDS-PAGE. The markers contain 10 proteins with exact masses of 5 kda, 10 kda, 15 kda, 25 kda, 35 kda, 50 kda, 75 kda, 100 kda, 150 kda, and 225 kda. The 50 kda band is double-intensity for easy identification. Simple Wide distribution of exact masses simplifies sample determination Accurate Recombinant proteins do not contain oligosaccharides that can cause anomalous migration, heterogeneous fuzzy bands, and inaccurate size estimation Performance of ProSieve Protein Markers in various gel concentrations kda % 12% 10% 7.5% 10-20% 4-20% 8-16% 4-12% Versatile Before Western blotting, markers can be visualized in gel with SYPRO Tangerine Gel Stain (page 55) without inhibition of protein transfer Storage Conditions -20 C Ordering Information ProSieve Protein Marker Cat. No. Size Price μl $ Gels were run at 175 volts until the dye front reached the bottom of the gel (approx. 60 minutes). 8 μl 10 μl of marker was loaded per lane (0.8 μg 1 μg per band). Gels were stained with Coomassie Brilliant Blue Stain. AccuGENE Electrophoresis Buffers Optimum performance AccuGENE Electrophoresis Buffers are formulated to match PAGEr Precast Gels. AccuGENE Buffers for protein electrophoresis are prepared with high quality reagents and use 18 megohm water. Products are filtered using a 0.2-micron filter. Reliable Manufactured according to strict quality control standards to ensure lot-to-lot consistency E cient Ready-to-use solutions eliminate preparation time Flexible Customized solutions are available to meet individual needs Storage Conditions 18 C 24 C Ordering Information Buffers for Protein Electrophoresis Cat. No. Size Price AccuGENE 10X Tris-Glycine Buffer 0.25 M Tris base, 1.92 M Glycine L $ L $81 AccuGENE 10X Tris-Glycine SDS Buffer 0.25 M Tris base, 1.92 M Glycine, 1% SDS L $ L $81 Related Products PAGEr Minigel Chamber 50 PAGEr Precast Gels 49 ProSieve 50 Acrylamide Gel Solution 60 52

61 SYPRO Protein Gel Stains Fast, sensitive, easy-to-use protein gel stains SYPRO Protein Gel Stains are simple, sensitive alternatives to Coomassie Brilliant Blue and silver stain for a diverse range of applications from 2D gel staining to staining gels prior to Western blotting. Exquisitely sensitive Detection limits rival the best silver stains Fast and easy Simple procedures require no complex fixation or destain Quantitative Broad linear range over 3 orders of magnitude Versatile Visualize with UV transilluminators, Dark Reader Transilluminators, and laser scanners Compatible With downstream processing such as mass spectrometry and microsequencing Sensitivity of SYPRO Stains compared to Coomassie Brilliant Blue and silver stain Select the Best Stain for your Application Application SYPRO Ruby SYPRO Tangerine SYPRO Red High performance staining Staining prior to Western blotting 2D Electrophoresis Edman microsequencing Mass spectrometry Quantitation Zymography Electroelution Membrane staining Protein expression Detection prior to Immunostaining Di cult to stain proteins IEF Gels Protein Markers, Buffers, and Stains Serial dilutions of ProSieve Protein Marker 50 kda band on 12% PAGEr Gold Precast Gels, stained and photographed as noted. Protein levels indicated in nanograms. Coomassie Brilliant Blue Stain (CCD camera) Fast, simple staining procedure SYPRO Red Protein Gel Stain diluted in 7.5% acetic acid (Polaroid Photo UV light) Stain Wash Fixation is required for staining 2D gels in SYPRO Ruby Gel Stain. No wash step is necessary for SYPRO Red or Tangerine Gel Stains. SYPRO Tangerine Protein Gel Stain diluted in PBS (Polaroid Photo UV light) Silver Stain (Amersham Plus One Kit) Application Support Detection of Proteins in Polyacrylamide Gels, Section X, page 166 SYPRO Protein Gel Stains, Section X, page 167 SYPRO Ruby Stain Related Products PAGEr Minigel Chamber 50 PAGEr Precast Gels 49 ProSieve 50 Acrylamide Gel Solution 60 ProSieve Protein Markers 52 53

62 SYPRO Ruby Protein Gel Stain Protein Markers, Buffers, and Stains The best stain for 2D gel analysis SYPRO Ruby Protein Gel Stain is a highly sensitive, simple to use fluorescent protein gel stain that can accurately quantitate protein expression levels and is compatible with standard fluorescent visualization systems and downstream identification techniques such as mass spectrometry. Highly sensitive Rivals the best silver stain Quantitative Broad linear range and consistent gel-togel staining Fast Simple staining procedure saves time and money High throughput Fast, easy staining of multiple gels Versatile Detects di cult to stain proteins Storage Conditions 18 C 26 C Ordering Information Cat. No. Size Price SYPRO Ruby Protein Gel Stain SYPRO Ruby Stain is supplied in a ready-to-use, single reagent format. The 1 L size stains approximately 20 minigels or 2 large 2D gels. The 5 L size is provided in a box with a spigot for dispensing ml $ L $ L $964 Product licensed from Molecular Probes, Inc. Photographic filter recommended (page 55) SYPRO Ruby vs. Silver Stain for 2D Analysis SYPRO Red Protein Gel Stain The fastest, easiest stain for detecting proteins SYPRO Red Protein Gel Stain is a fast, highly sensitive fluorescent protein gel stain that detects as little as 4 ng 8 ng protein per band. Fast Complete staining in less than 1 hour Sensitive Five times more sensitive than Coomassie Brilliant Blue stain Simple No fixation or destaining required Consistent Low protein-to-protein variability Staining is easy simply soak gels in a solution of 1X SYPRO Red Stain in 7.5% acetic acid for 40 to 60 minutes. The stain is compatible with UV transilluminators, CCD cameras or laser scanners. Storage Conditions 18 C 26 C Proteins from a cell lysate were run on a 2D gel and stained with SYPRO Ruby Gel Stain (left) or silver stain (right) Ordering Information Cat. No. Size Price SYPRO Red Protein Gel Stain SYPRO Red Stain is supplied as μl or 500 μl as a 5,000X concentrate, su cient for staining approximately 50 minigels μl $ μl $168 Product licensed from Molecular Probes, Inc. Photographic filter recommended (see page 55) SYPRO Red Gel Stain SDS Polyacrylamide gel stained with SYPRO Red Gel Stain 54

63 SYPRO Tangerine Protein Gel Stain Ideal for staining gels prior to Western blotting SYPRO Tangerine Protein Gel Stain is a versatile, sensitive stain that can be used to visualize proteins prior to Western blotting. Visualize proteins prior to transfer Does not interfere with protein activity or transfer Safe No acids or organic solvents necessary Sensitive Detects as little as 4 ng 8 ng protein per band The staining procedure is fast and simple and does not require the use of organic solvents; staining can be performed in saline or PBS solutions. Proteins can be used in zymography assays or analyzed by mass spectrometry. Storage Conditions 18 C 26 C SYPRO Ruby Protein Blot Stain Ordering Information Cat. No. Size Price SYPRO Tangerine Protein Gel Stain Supplied as a 5,000X concentrated solution in DMSO, su cient for staining 50 minigels μl $162 Product licensed from Molecular Probes, Inc. Performance of SYPRO Tangerine Gel Stain A. B. Two identical SDS-PAGE gels were run with samples of protein molecular weight standards (leftmost lanes) and protein molecular weight standards mixed with decreasing amounts of E. coli -glucuronidase and rabbit liver esterase. Gels were stained for total protein with SYPRO Tangerine Protein Gel Stain, and for specific enzymatic activities. Both gels were first stained with SYPRO Tangerine Protein Gel Stain (one gel shown, Panel A). One gel was stained with ELF -97 -d-glucuronidase substrate (E-6587) for the detection of -glucuronidase activity (Panel B). Protein Markers, Buffers, and Stains Fast, simple, sensitive stain for detecting proteins on blots SYPRO Ruby Protein Blot Stain offers sensitivity levels that rival colloidal stains. The stain is 60-times more sensitive than reversible stains like Ponceau S, and 30-times more sensitive than Amido Black or Coomassie Brilliant Blue Stains. Highly Sensitive Detects as little as 2 ng 8 ng protein per band Fast Simple staining procedure takes less than 1 hour Compatible With fluorogenic, chemiluminescent and colorimetric detection techniques Storage Conditions 18 C 26 C SYPRO Protein Gel Stain Photographic Filter Ordering Information Cat. No. Size Price SYPRO Ruby Protein Blot Stain ml $136 Product licensed from Molecular Probes, Inc. Total protein detection with SYPRO Ruby Protein Blot Stain Molecular weight standards containing decreasing amounts of -tubulin were run on an SDS-PAGE gel, blotted onto a PVDF membrane and stained with SYPRO Ruby Protein Blot Stain. For optimal detection sensitivity with black and white film photography The SYPRO Protein Gel Stain Photographic Filter is suitable for Polaroid Camera Systems. The filter does not work with CCD camera systems. Check with the manufacturer for the appropriate filter. Recommended for use with all SYPRO Protein Gel Stains. Ordering Information Cat. No. Size Price SYPRO Protein Gel Stain Photographic Filter inch square $57 (Wratten #9 Gelatin Filter) 55

64 IsoGel Agarose and Precast IsoGel Agarose IEF Plates IsoGel Products for Isoeletric Focusing Isoelectric focusing for rapid separation of large proteins Separation of proteins in complex mixtures for analytical resolution can be achieved by isoelectric focusing (IEF), in which proteins are separated based on their net charge (isoelectric point or pi) in the presence of a ph gradient. Agarose has distinct advantages over polyacrylamide gels for isoelectric focusing. Separation in agarose is more rapid, and agarose gels can be used to separate proteins up to 2,000 kda. Lonza has developed two high quality products that are specifically designed and tested for their performance with IEF. IsoGel Agarose is a highly purified agarose that is easy to prepare and produces a gel with high clarity and a less restrictive matrix than polyacrylamide IsoGel Agarose IEF Plates are ready-to-use precast gels supported on GelBond PAG Support Film, eliminating gel preparation time and providing easy handling throughout the IEF process Advantages Safe No toxic acrylamide required Fast Shorter staining times Simple Nontacky and easy to blot Applications Isoelectric focusing Antibody separation and analysis Immunofixation directly in the gel Crossed immunoelectric focusing Direct tissue or preparative isoelectric focusing Protein blotting Immunodetection of proteins Application Support Isoelectric Focusing of Proteins on Agraose Gels, Section XII, page 177 IsoGel Agarose Highly purified agarose for isoelectric focusing No measurable EEO Manufacturing process minimizes fixed anions and mobile cations Versatile Su ciently rigid for casting in vertical tubes (e.g. O Farrell gels 1 ), vertically molded or horizontally open cast thin gels Applications Isoelectric focusing Analytical Specifications Moisture: 10% Sulfate: 0.20% EEO (m r ): Not detectable Gel strength (1.5%): 500 g/cm 2 IEF test: Passes test Storage Conditions 18 C 26 C Reference O Farrell, P.H. (1975) High resolution two-dimensional electrophoresis of proteins. J. Biol. Chem. 250: Ordering Information Cat. No. Size Price IsoGel Agarose g $368 Larger package sizes are available upon request. Please inquire for pricing and availability. Related Products GelBond PAG Support Film 42 56

65 Precast IsoGel Agarose IEF Plates Precast gels for the analysis of antibodies and proteins up to 2,000 kda Easy handling Each gel is supported on GelBond PAG Support Film to provide dimensional stability throughout IEF processing Versatile Convenient 125 mm 100 mm gel size fits most horizontal IEF chambers Fast Proteins can be quickly transferred from gel to membrane, stained in situ, or detected by antibodies within 1 hour Applications Isoelectric focusing Antibody separation and analysis Performance and Quality Tests Each lot of IsoGel Agarose IEF Plates is functionally tested; Certificate of Analysis available upon request Storage Conditions 2 C 8 C for 12 months from the date of manufacture Accessories: 18 C 26 C Ordering Information Cat. No. Description Quantity Price Precast IsoGel Agarose IEF Plates Precast IsoGel Agarose IEF Plates ph range Precast IsoGel Agarose IEF Plates ph range Precast IsoGel Agarose IEF Plates ph range Precast IsoGel Agarose IEF Plate Accessory Pack Contains masks, 100 mm and 125 mm wicks and blotting paper for 6 plates Precast IsoGel Agarose IEF Plate Accessory Bulk Pack Contains 125 mm wicks and blotting paper Precast IsoGel Agarose IEF Blotting Paper 6 plates $269 6 plates $280 6 plates $280 Su cient for 6 plates $ each $ sheets $222 IsoGel Products for Isoeletric Focusing Performance of IsoGel Agarose IEF Plate Proteins Lentil lectin pi Equine myoglobin 7.0 Bovine carbonic anhydrase Ovalbumin 3.6 Amyloglucosidase Separation of proteins in an IsoGel Agarose IEF Plate, ph Lanes 1 & 4: pi Marker (in-house). Lanes 2 & 3: Broad Range pi marker (Bio-Rad ). Lane 5: Hemoglobin, HB Type AFSC (PE Wallac). 2.5 μl of each sample were loaded on the gel and prefocused at 1 watt for 10 minutes and focused at 2000 volts (max), 25 ma (max), 25 W (max) for 60 minutes on a GE MultiPhor II Chamber at 10 C. The gel was stained with Crowle s stain. Related Products GelBond PAG Support Film 42 57

66 Agarose for Protein Separation Agarose for Protein Separation Safe and easy separation of large proteins and protein complexes Electrophoresis of proteins in agarose gels has distinct advantages compared to polyacrylamide for some applications. Agarose gels can easily and effectively separate high molecular weight proteins and protein complexes (>600 kda). Safe No toxic monomer solutions required E cient recovery High recovery yields with simple procedures Flexible Gels can be made with standard Laemmli buffer systems Normal distribution of von Willebrand Factor multimers as separated on 1%, 2% or 3% SeaKem HGT(P) Agarose gels 278 kda subunits (n) kda Reference Warren, C.M. et al. (2003) Vertical agarose gel electrophoresis and electroblotting of high molecular weight proteins, Electrophoreis 24(11): Smejkal, G.B. et al. (2003) Rapid high-resolution electrophoresis of multimeric von Willebrand Factor using a thermopiloted gel apparatus. Electrophoresis 2003, 24: Agarose Concentration (%) vwf is the largest protein in the human plasma, the basic repeating unit of which is a 556 kda homodimer comprised of identical 278 kda subunits disulfide-linked at the C-termini. Each band subfractionates into three bands at higher gel concentrations. Gels prepared with 200 mm Tris, 100 mm glycine ph 9.0 supplementing with 0.1% LiDS. Gels run for 20 min at 10 C using 250 V. Proteins were transferred to nitrocellulose membranes and sequentially probed with anti-human vwf antisera and alkaline phosphatase secondary antibody and visualized using an enhanced NBT- BCIP substrate. Used by permission from Gary B. Smejkal, Cleveland State University, Cleveland, Ohio. Routine Protein Separation Agarose Typical Application Protein Size Range (kda) Gel Concentrationn MetaPhor Agarose Protein electrophoresis SeaKem Gold Agarose or SeaPlaque Agarose Specialty Protein Separation Protein electrophoresis 600 1,000 1,000 5,000 IsoGel Agarose Isoelectric focusing Separation based on isoelectric point SeaKem HGT Agarose Counter-Immunoelectrophoresis (CIEP Crossed-IEP SeaKem HGT(P) Agarose Factor VIII/von Willenbrand s factor multimer separation SeaKem ME Agarose Serum protein electrophoresis SeaKem HEEO Agarose Immunoelectrophoresis of IgG and IgM SeaKem HE Agarose Serum protein electrophoresis, IEP, Crossed-IEP,CIEP 4% 3% 2% 1.5% 1% 58

67 Agarose for Protein Separation Continued Ordering Information Cat. No. Size Price SeaKem HGT Agarose High gel strength, high clarity agarose for use in counter-immunoelectrophoresis and crossed immunoelectrophoresis g $ g $281 SeaKem HGT(P) Agarose Used for separating Factor VIII/von Willenbrand s (VWF) factor multimers g $753 SeaKem HE Agarose A high EEO agarose that provides enhanced resolution in immunoelectrophoresis, crossed-immunoelectrophoresis, counter-immunoelectrophoresis, and serum protein electrophoresis g $ g $250 SeaKem HEEO Agarose A very high EEO agarose useful in applications requiring significant cathodal migration, such as immunoelectrophoresis of IgG and IgM. May also be blended with lower EEO agarose to achieve a specific EEO value g $ g $327 SeaKem ME Agarose An ideal alternative to polyacrylamide for serum protein electrophoresis g $ g $ g $627 Ordering Information Cat. No. Size Price MetaPhor Agarose Effective for separating proteins 600 kda g $ g $ g $1,287 Larger package sizes are available upon request. Please inquire for pricing and availability. SeaKem Gold Agarose Effective for separating proteins 600 kda g $ g $552 Larger package sizes are available upon request. Please inquire for pricing and availability. SeaPlaque Agarose A low melting alternative for separation of proteins 600 kda g $ g $436 IsoGel Agarose For use in isoelectric focusing g $368 Agarose for Protein Separation Larger package sizes are available upon request. Please inquire for pricing and availability. Analytical specifications SeaKem HGT SeaKem HGT(P) SeaKem HE SeaKem HEEO SeaKem ME Gelling temp. (1.5%): 42 C ± 1.5 C 42 C ± 1.5 C 36 C ± 1.5 C 36 ± 1.5 C 36 ± 1.5 C Moisture: 10% 10% 10% 10% 10% Sulfate: 0.30% 0.20% 0.20% 0.25% 0.20% EEO (mr): Gel strength (1%): 800 g/cm 2 1,000 g/cm g/cm g/cm 2 1,000 g/cm 2 Storage Conditions 18 C 26 C Application Support Protein Separation in Agarose Gels, Section XIII, page 195 Related Products PAGE AccuGENE Buffers 52 GelBond PAG Support Film 42 Precast IsoGel Agarose IEF Plates 56 59

68 ProSieve 50 Acrylamide Gel Solution ProSieve 50 Acrylamide Gel Solution Modified acrylamide formulation for high performance electrophoresis of large proteins Gradient separation From easy-to-cast single concentration gels Easy-to-handle Gels are more durable than standard acrylamide Sharp resolution Resolves large proteins (>200 kda) Fast Shorter destaining times and faster protein mobility times Low Background Even when used with silver stain Ordering Information Cat. No. Size Price ConduitITCStd (50% concentrate) ml $ ml $202 Applications Protein gel electrophoresis Storage Conditions 18 C 26 C Application Support Protein Separation in Polyacrylamide Gels, Section X, page 155 Performance of ProSieve 50 Acrylamide Gel Solution 200 kda 10% ProSieve 50 Gel % gradient polyacrylamide gel kda 10 kda ProSieve Protein Markers were loaded in lanes 1, 5, and 10; aqueous extract from E. coli bacterial cells was loaded in lanes 2, 6, and 9; Protein Markers, broad range were loaded in lanes 3 and 7; and 10 kda Protein Ladder was loaded in lanes 4 and 8. Proteins were detected by Coomassie Brilliant Blue Stain. Related Products 60 GelBond PAG Support Film 42 AccuGENE Buffers 41

69 GelBond PAG Support Film Polyacrylamide support film GelBond PAG Support Film is a transparent, flexible polyester film designed to support polyacrylamide or MDE Gels. The acrylamide monomers covalently attach to the coating on the film during the polymerization reaction. Gels remain permanently attached to the film through electrophoresis and all subsequent fixing, staining, destaining, and drying procedures. Reliable Polyacrylamide gels retain their original dimensions during staining and after drying Durable Gels, particularly thin ones, are easier to handle during staining, destaining, and drying when supported Convenient Gel orientation can be recorded directly on the GelBond PAG Support Film prior to casting NOTE: Polyester films will not transmit light of less than 310 nm, and will fluoresce at higher wavelengths. Applications Drying and support of polyacrylamide gels Storage Conditions 18 C 26 C, protect from light Ordering Information Cat. No. Sheet Size (mm) Chamber Compatibility Quantity (sheets) Price GelBond PAG Support Film Sheets GelBond PAG Support Film sheets for polyacrylamide gels; 0.2 mm thick sheets $ Hoefer SE400, SE600, 50 $289 Bio-Rad PROTEAN II GE Multiphor 50 $ $ $ GE Multiphor II 50 $ Biometra SA $ X-ray size 10 $289 Custom-cut GelBond PAG Support Film is available on special request. Please inquire for pricing and availability. GelBond PAG Gel Support Film GelBond PAG Support Film Related Products AccuGENE Buffers 41 ProSieve Protein Markers 51 SYPRO Protein Stains 53 MDE Solution 44 61

70 Notes 62

71 SourceBook Chapter 3

72 Notes 64

73 Preface Preface Biolog ochemistr lecular Biolog nomics nctional Genomic oteomics The names used to define areas of life science research are continually changing, but many of the basic questions researchers ask remain the same. Many of the techniques used remain the same techniques that have been applied for many years. Separation of nucleic acids and proteins by gel electrophoresis remains a fundamental tool of life science research, and Lonza continues to drive innovations that make this work faster, easier and more reliable. Marine Colloid C BioProduct owhittaker Molecular Application mbre nza Our company name has also changed over the years. But our high quality products and our commitment to our customers have not. For over four decades, we have developed and supplied the research community with the best products in the industry for separation, detection, modification and recovery of nucleic acids and proteins. We have compiled those years of experience into a new edition of The Sourcebook. In the following pages, you will find reference information and techniques to help you get maximum performance from our products, as well as your electrophoresis applications in general. We hope that you like our products and this manual becomes one of your preferred sources for all your electrophoresis needs. We welcome your comments. Please contact us with your feedback at: scientific.support.eu@lonza.com 65

74 Table of Contents Table of Contents I. Frequently Asked Questions Nucleic Acid FAQs 70 Protein Analysis FAQs 72 II. Preparation of Agarose Gels Agarose Selection Guide 76 Agarose and Compatible Techniques 76 Agarose Types 77 Agarose Analytical Specifications 79 Suggested Agarose Concentrations and Dye Migration Information 80 Buffers for Electrophoresis 81 Dissolving Agarose 83 Casting Agarose Gels 85 III. Loading and Running DNA in Agarose Gels DNA Loading 90 Loading Buffers 91 Optimal Voltage and Electrophoretic Times 92 Fast Running Protocols for High Resolution in MetaPhor Agarose Gels 93 References 94 IV. Detection and Sizing of DNA in Agarose Gels Guide to Lonza Ladders and Markers 96 Detecting DNA with GelStar and SYBR Green Nucleic Acid Gel Stains 98 Detecting DNA with Ethidium Bromide 102 High Sensitivity Detection Using the FlashGel System 103 References 104 V. In-Gel Reactions Overview 106 Tips for Increasing the Efficiency of In-Gel Reactions 107 Cloning in the Presence of Agarose 108 Restriction Digestion in the Presence of Agarose 112 DNA Amplification in the Presence of Agarose 113 References 113 VI. Recovery of DNA from Agarose Gels Tips for Increasing DNA Recovery Efficiency from Agarose Gels 116 ß-Agarase Recovery of DNA from Agarose Gels 117 Electroelution of DNA from Agarose Gels 126 Phenol/Chloroform Extraction of DNA from Agarose Gels 122 Modified Freeze/Squeeze Extraction of DNA from Agarose Gels 123 Ethanol Precipitation of DNA Recovered from Agarose Gels 124 References 124 VII. Separation of DNA in Polyacrylamide Gels Separating DNA in Polyacrylamide Gels 126 Detecting DNA in Polyacrylamide Gels 127 Recovery of DNA in Polyacrylamide Gels 129 References 130 VIII. Separation of RNA in Agarose Gels Introduction 132 Preparation of RNA Samples 132 Buffers for RNA Electrophoresis 134 Electrophoresis of RNA 134 Detection of RNA in Agarose Gels 138 Detecting RNA with Ethidium Bromide 142 Northern Blotting 143 FlashGel Systems for RNA Analysis 146 References 145 IX. Special Applications in Agarose Gels Amplification of Plasmid cdna Libraries with SeaPrep Agarose 150 Preparing Agarose for use in Cell Culture Applications 152 References

75 Table of Contents Table of Contents continued X. Protein Separation in Polyacrylamide Gels Buffers for Protein Electrophoresis 157 Casting Polyacrylamide Gels 157 Loading and Running Proteins on Polyacrylamide Gels 159 Loading Buffers 160 Optimal Voltage, Running Times and Power Settings 161 ProSieve 50 Gel Solution 161 PAGEr Gold Precast Polyacrylamide Minigels 162 Chamber Modification Instructions 164 Detection of Proteins in Polyacrylamide Gels 166 SYPRO Protein Gel Stains 166 Coomassie Blue Stain 168 Silver Stain 169 References 170 XI. Blotting Proteins from Polyacrylamide Gels Introduction 172 Semi-dry Blotting PAGEr Gold Precast Gels 173 Tank Blotting PAGEr Gold Precast Gels 174 References 175 XII. Isoelectric Focusing of Proteins on Agarose Gels Introduction 178 Preparation of Isoelectric Focusing Gels 179 Running Agarose IEF Gels 182 Staining Proteins in Agarose IEF Gels 184 Press Blot Transfer of Proteins from Agarose IEF Gels 188 Preparative Isoelectric Focusing 189 Immunofixation/Immunoperoxidase or Autoradiography 190 Direct Tissue Isoelectric Focusing 191 Resolution Reference Guide 192 References 194 XIII. Protein Separation in Agarose Gels Introduction 196 Buffers for Protein Separation in Agarose 197 Casting Agarose Gels for Protein Separation 198 Preparation and Loading of Protein Samples 198 Optimal Voltage and Electrophoretic Times 199 Detection of Proteins in Agarose Gels 199 Gel Drying and Preservation 200 Processing Agarose Gels Following Electrophoresis 200 Recovering Proteins from Agarose Gels 201 References 202 Appendix A. Electrophoretic Theory Voltage, Current and Power; Interactive Effects on Gel Electrophoresis 204 Electrophoretic Parameters 205 Appendix B. Agarose Physical Chemistry AgarosePhysical Chemistry 208 References 209 Appendix C: Safety Safety and Environmental Precautions 210 Table of Contents 67

76 Notes 68

77 Section I: Frequently Asked Questions FAQs In This Section Nucleic Acid FAQs 70 Protein Analysis FAQs 72 69

78 Section I: Frequently Asked Questions Nucleic Acid FAQs Nucleic Acid FAQs Electrophoresis and Agarose Q. What buffer conditions give me best resolution for agarose electrophoresis? A. For small DNA fragments (<1,000 bp) when recovery is not necessary, we recommend the use of 1X TBE Buffer. For any given concentration of agarose, gels made with TBE Buffer give sharper bands than gels made with TAE Buffer. TBE results in better resolution for closely spaced DNA bands. For large DNA fragments (>15,000 bp), 1X TAE Buffer enhances separation of large DNA. Since TAE has a lower buffering capacity, it may be necessary either to recirculate the buffer, or periodically mix the buffer between the anodal and cathodal chambers when electrophoresing for an extended period of time. The time to buffer depletion can vary with the volts/hour and the size of chamber used. Whichever buffer you use, the depth over the gel should be 3 to 5 mm deep. Less buffer, and you risk the chance of the gel drying out. Excessive buffer will decrease the resistance of the circuit between the anode and cathode, which results in a decreased voltage gradient through the gel. This causes inefficient DNA mobility, excessive heating, and band distortion. Q. How should I cast my gels to get the best resolution? A. The first concern is the thickness of the gel. We usually cast gels 3 mm to 4 mm thick. The gel volume needed can easily be estimated by measuring the surface area of the casting chamber, then multiplying by the gel thickness. Thinner gels can be cast on GelBond Support Film, and/or cast in a vertical apparatus. The thickness of the comb in the direction of the electrical field can also profoundly affect the resolution. A thin comb (1 mm) will result in sharper DNA bands. With too thick a comb, the separated DNA bands will be quite broad. Q. My DNA bands are sometimes wavy, but usually only in one or two lanes. What causes this? A. Dried agarose on the comb teeth is a frequent cause of this problem. Prior to casting your gel, check the comb teeth for residual dried agarose. If not removed, this will attach to the newly cast agarose and fracture the well upon comb removal. This is usually not observable until the gel is on the transilluminator. Additionally, care must be taken during comb removal, particularly with low melting temperature agaroses. Well integrity may be maintained in these agaroses by pre-chilling the gel to 4 C for 30 minutes and/or by ooding the gel with cold buffer prior to removing the comb. Q. How much DNA should I load per well? A. The amount to load per well is variable. What is most important is how much DNA there is in the bands you wish to resolve. The least amount of DNA that can be consistently detected with ethidium bromide is about 10 ng. The most DNA you can have in a band and still get a sharp, clean band on an ethidium bromide stained gel is about 100 ng. These amounts will be less on gels stained with more sensitive stains such as GelStar Stain. On a GelStar Stained Gel it is possible to detect as little as 20 pg dsdna. The optimal amount of DNA to load in the well is calculated by the fraction of the total DNA which is in the band of interest. If you are unsure of how much DNA is present, load varying amounts in several lanes if possible. To further increase the sharpness of the bands, use a Ficoll based loading buffer such as Lonza DNA Loading Buffer (Cat. No ) instead of sucrose-based or glycerol-based loading buffers. The use of lower molecular weight glycerol will allow DNA to stream up the sides of the well before electrophoresis which results in U-shaped bands. Loading buffer that is too high in ionic strength can cause the bands to be fuzzy. In the ideal situation, the DNA sample should be suspended in the same solution as the running buffer. If this is not possible, use a sample buffer with a lower ionic strength than the running buffer. 70

79 Section I: Frequently Asked Questions Nucleic Acid FAQs continued Electrophoresis and Agarose Q. At what voltage should I run an agarose gel? A. We recommend running agarose gels at 4 10 volts/cm (cm is determined by measuring the interelectrode distance, not the gel length) under normal horizontal electro phoretic conditions. If the voltage is too high, band streaking, especially for DNA >15 kb, may result. When the voltage is too low, the mobility of small (<1,000 bp) DNA is reduced and band broadening will occur due to diffusion. MetaPhor Agarose gels separate DNA optimally at volts /cm in standard horizontal electrophoresis systems. Higher voltages result in a decrease in the resolution of DNA separation, mainly due to gel overheating. Another special case is the separation of large (>15 kb) DNA fragments using conventional horizontal electro phoresis. The best separations in this instance are obtained at a voltage gradient of <5 volts /cm. Q. What is the difference between NuSieve 3:1 and NuSieve GTG Agaroses? A. NuSieve 3:1 Agarose is a standard melting temperature agarose. The resolution range for NuSieve 3:1 Agarose is 50 bp 1000 bp. NuSieve 3:1 Agarose is designed for analytical electrophoresis; its high gel strength also makes it ideal for use in various blotting techniques. NuSieve GTG Agarose is a low melting temperature agarose ( 65 C at 4%). The resolution range for this agarose is 50 bp 1000 bp. NuSieve GTG Agarose is recommended for in-gel applications such as cloning or ligation and transformation. GTG stands for Genetic Technology Grade. GTG Grade Agarose is recommended for preparative DNA electrophoresis, or when further enzymatic manipulation of DNA is required. These agaroses are extensively tested to ensure maximum compatibility with standard molecular biology techniques. Q. What is the difference between SeaKem LE and SeaKem GTG Agaroses? A. SeaKem LE and SeaKem GTG Agaroses are both standard melting temperature agaroses. The resolution range for these agaroses is 100 bp to 23,000 bp. SeaKem LE Agarose is ideal for routine analysis of DNA. SeaKem GTG Agarose is a Genetic Technology Grade Agarose, specifically designed for preparative DNA electrophoresis. Q. What is the difference between SeaPlaque and SeaPlaque GTG Agaroses? A. SeaPlaque and SeaPlaque GTG Agaroses are both low melting temperature agaroses ( 65 C at 1.5%). The resolution range for these agaroses is 200 bp 25,000 bp. SeaPlaque Agarose is recommended for pre parative DNA electrophoresis. SeaPlaque GTG Agarose is a Genetic Technology Grade Agarose, recommended for direct enzymatic manipulation of nucleic acids in remelted agarose (in-gel reactions). It is also compatible with PCR and sequencing reactions carried out in the presence of the remelted gel. Precast Agarose Gels Q. Do I need to purchase a special chamber to use Reliant and Latitude Precast Gels? A. Reliant and Latitude Precast Agarose Gels are designed to run in standard horizontal electrophoresis chambers. As long as there is room on the chamber platform for the gel, the chamber should be suitable. Measure the chamber platform and check against precast gel size to be sure. For example, the OWL Centipede Chamber is ideal with the 14 cm 24 cm Latitude HT Precast Gels; the OWL B1 EasyCast Chamber is good with Reliant Precast Agarose Gels; and the Latitude Chamber is perfect with Latitude Midigels. Results with different chambers will vary depending on differences in chamber size and construction. Q. Can I use the FlashGel System for both DNA and RNA? A. Yes. We offer FlashGel Cassettes and Reagents for both DNA and RNA. The FlashGel Dock may be used for both cassette types as there is no risk of contamination. Q. Are the materials in the FlashGel Cassette hazardous? A. The stain in the FlashGel Cassette is present at such low levels that it is not considered hazardous according to OSHA and EU hazard criteria. A copy of the MSDS is available online. The stain in the cassette is a potential mutagen. Wear gloves, safety glasses and a lab coat when handling. Use the same precautions when handling and disposing of the cassettes as you would ethidium bromide stained gels. Nucleic Acid FAQs 71

80 Section I: Frequently Asked Questions Protein Analysis FAQs Protein Analysis FAQs PAGEr Precast Gels Q. Which PAGEr Precast Gels will fit my gel chamber? A. PAGEr Precast Gels are available in 9 cm 10 cm and 10 cm 10 cm sizes and fit most standard mini-vertical systems. Some chambers may require modifications for optimal fit with PAGEr Precast Gels. Standard Vertical Systems PAGEr Gels PAGEr Minigel Chamber 9 cm 10 cm 10 cm 10 cm gels Bio-Rad MiniPROTEAN II, MiniPROTEAN 3 or 9 cm 10 cm gels Ready Gel Cell Systems Reverse the inner core gasket so the at side faces outward. Novex XCell SureLock Mini-Cell 9 cm 10 cm 10 cm 10 cm gels Request the spacer for the XCell SureLock, Mini-Cell Chamber from Scientific Support, (Cat. No ). FisherBiotech Vertical Minigel FBVE121, 10 cm 10 cm gels Owl Separations Systems Wolverine P82 Chamber comes with 2 sets of wedges. Use the thinner wedges for the PAGEr Gold Gels. FisherBiotech Vertical Minigel FB-VE101, 10 cm 10 cm gels Owl Separations Systems Penguin Model P8DS Request adaptor for these chambers from Scientific Support, (Cat. No ). Hoefer Mighty Small (SE250) 9 cm 10 cm 10 cm 10 cm gels Replace the buffer chamber with a Deep lower buffer chamber for the SE260, order number , from GE Healthcare. Daiichi 2, ISS chambers 10 cm 10 cm gels To run one gel: Place one cm cassette on wedge side of chamber. Use suitable buffer dam on the other side. Use regular Daiichi/ISS wedges. To run two gels: Widen the hole on the yellow port of the inner core. Replace the long arm wedges with modified wedges.this chamber modification and new wedges are available from Scientific Support. Novex XCell II Hoefer Mighty Small (SE260) EC 120 Mini Vertical Gel System Biometra Mini V Chamber CBS Scientific MGV System, (10 cm 8 cm units) Sigma-Aldrich Mini Techware (11.3 cm 10 cm units) Zaxis System 2000 Hoefer Mini VE 9 cm 10 cm or 10 cm 10 cm gels 9 cm 10 cm or 10 cm 10 cm gels 9 cm 10 cm or 10 cm 10 cm gels 9 cm 10 cm gels 9 cm 10 cm gels 10 cm 10 cm gels 10 cm 10 cm gels 10 cm 10 cm gels Q: Do Lonza PAGEr Precast Gels contain a stacking gel? What is the purpose of the stacking gel? A. PAGEr Gold Precast Gels contain a 4% stacking gel, ph 8.6. The purpose of this stacking gel is to allow the proteins to accumulate and condense (i.e. stack) at the stacking/resolving gel boundary. This stacking effect results in superior resolution within the running gel. Q. I would like to run a native or nondenaturing gel. What can I use? A. PAGEr Precast Gels do not contain SDS or any other denaturing agents (e.g. DTT and -ME). Additionally, you would use a Tris-Glycine Running Buffer that does not contain SDS. Protein Electrophoresis Q. How do I make the transfer, running, and sample buffers? A. Tris-Glycine Gels (Tris-HCl Buffer System) Towbin Transfer Buffer (1X) Running Buffer (1X) Sample Buffer (1X) M Tris base 25 mm Tris Base 62.5 mm Tris-HCl, ph M Glycine 192 mm Glycine 2% SDS* % SDS* 0.1% SDS* 10% Glycerol 20% Methanol 0.01% Bromophenol Blue 2.5% ME (2-mercaptoethanol)* *Omit for native proteins For best results use Lonza AccuGENE Electrophoresis Buffers Q. What is the difference between gradient vs. homogeneous (single concentration) gels? Which one should I use? A. Gradient gels are suitable for a wide range of size resolutions. A homogeneous or single concentration gel is appropriate where the proteins of interest are known to be within a narrow size range. Q. How much protein should I load on the gel? A. Protein load levels will vary depending upon sample purity and staining method used. For highly purified proteins, 0.5 g to 5 g protein per lane on a minigel is generally sufficient. Complete mixtures such as cell lysates may require as much as 50 g protein per lane. Protein stain detection limits Lower Detection Limit Protein Stain (Protein / Band) Coomassie Blue Stain 30 ng Silver Stain 2 ng SYPRO Red Protein Gel Stain 4 ng 8 ng SYPRO Ruby Protein Gel Stain 2 ng 8 ng SYPRO Tangerine Protein Gel Stain 4 ng 8 ng NOTE: Limits are based on optimal detection methods for each stain. 72

81 Section I: Frequently Asked Questions Protein Analysis FAQs continued Q. What is the best membrane to use for Western blotting? A. Use this table to find a suitable membrane. Nitrocellulose PVDF Nylon Hydrophobic binding Hydrophobic binding Hydrophobic and electrostatic binding General purpose SDS tolerant Stable if baked membrane Low background High background High background Low strength High strength High strength Becomes brittle if baked Q. What are the benefits of using agarose for protein gel electrophoresis? A. Protein electrophoresis in agarose gels is an alternative approach to using polyacrylamide gels and provides several benefits: Separate high molecular weight proteins ( > 600 kda) Easy to prepare and handle Efficient recovery of proteins Excised proteins can be used to immunize animals directly for antibody production Non-toxic Suitable for protein sequencing Least suitable for Western transfer Run gels using either a vertical or horizontal apparatus ProSieve Protein Markers Q. In what applications do you recommend using the ProSieve Color Protein Marker vs. the ProSieve Protein Marker? A. The ProSieve Color Protein Markers are ideal for monitoring protein separation during electrophoresis and confirming protein transfer in Western blotting. They are not recommended for precise sizing of protein samples in SDS-PAGE. The ProSieve Protein Markers are recommended for the most accurate sizing of protein samples in SDS-PAGE. These markers contain 10 proteins with exact masses of 5 kda, 10 kda, 15 kda, 25 kda, 35 kda, 50 kda, 75 kda, 100 kda, 150 kda, and 225 kda. Protein Analysis FAQs 73

82 Notes 74

83 Section II: Preparation of Agarose Gels Preparation of Agarose Gels In This Section Agarose Selection Guide 76 Agarose and Compatible Techniques 76 Agarose Types 77 Agarose Analytical Specifications 79 Suggested Agarose Concentrations & Dye Migration Information 80 Buffers for Electrophoresis 81 Dissolving Agarose 83 Casting Agarose Gels 85 75

84 Section II: Preparation of Agarose Gels Agarose Selection Guide Selecting the best agarose for your application can minimize opportunity for error, optimize results, and even reduce cost. Lonza offers a wide range of agarose types that are specifically engineered to optimize results by fragment size, sample type and application. The selection tools below will get you started. The following pages will guide you to the right concentration, buffer and marker to use for best performance in your experiment. Choose the Agarose that is Right for You 1 kb - 10 Mb GTG 50 bp - 1 kb GTG 200 bp - 25 kb 200 bp - 25 kb 100 bp - 23 kb 100 bp - 23 kb GTG GTG 50 bp - 1 kb Preparation of Agarose Gels 20 bp bp bp 400 bp 600 bp 800 bp 1,000 bp 25,000 bp Agarose and Compatible Techniques GTG 10,000,000 bp SeaKem SeaKem SeaPlaque SeaPlaque NuSieve NuSieve MetaPhor SeaKem SeaPrep InCert I.D. NA Recovery Method LE GTG GTG 3:1 GTG Gold In-Gel Reactions ß-Agarase Phenol/Chloroform Recovery Columns Electroelution Freeze/Squeeze Blotting Southern <1 kb Southern >1 kb Northern <1 kb Northern >1 kb Specialty Applications Viral Plaque Assays Preparation of Megabase Samples PFGE Cell Culture Encapsulation & Embedding of Cells DNA Identity Testing Comet Assays Our agarose is GUARANTEED DNase/RNase Free 76

85 Section II: Preparation of Agarose Gels Agarose Types Introduction Nucleic acid applications take advantage of the specific properties of different agaroses and derivatized agaroses. The appropriate choice of agarose depends on the size of the DNA to be analyzed and any subsequent manipulations required. Gelling/ melting temperatures, electroendosmosis and gel strength are all important factors in choosing the right agarose for your application. Refer to page 79 for analytical specifications of Lonza agarose. Genetic Technology Grade (GTG ) Agaroses Our Genetic Technology Grade (GTG ) Agarose Products are specially prepared and certified for demanding molecular biology applications for nucleic acids, including PCR amplified products. Lonza's GTG Agarose quality tests go beyond standard assays such as, DNase and RNase testing, to include enzymatic performance measurements. Our additional testing provides a more realistic index of overall product quality and reliability. You no longer need to screen agarose lots to find those that yield biologically active DNA. The following agaroses are GTG Certified: SeaKem GTG Agarose SeaPlaque GTG Agarose (low melting temperature agarose) NuSieve GTG Agarose (low melting temperature agarose) SeaKem Gold Agarose Lonza performs the following tests on GTG Certified Agaroses: DNA binding DNase and RNase activity DNA resolution Gel background-gel exhibits low background uorescence after ethidium bromide staining In-gel cloning (low melt agarose) In-gel restriction digestion (low melt agarose) Restriction-ligation assay (SeaKem GTG ) Molecular Biology Grade Agaroses Molecular biology grade agaroses are suitable for general analytical separation of DNA. The following agaroses are considered molecular biology grade agaroses: MetaPhor Agarose SeaKem LE Agarose NuSieve 3:1 Agarose SeaPlaque Agarose (low melting temperature agarose) Lonza screens our molecular biology grade agaroses for the following parameters: DNA binding DNase and RNase activity Gel background staining FDA Listing Lonza agarose types are listed as Class 1 Medical Devices under registration number Preparation of Agarose Gels 77

86 Section II: Preparation of Agarose Gels Agarose Types continued DNA resolution examples The photographs below show the different resolution properties of Lonza agaroses. NuSieve 3:1 A B NuSieve GTG A B MetaPhor A B SeaKem GTG A B SeaPlaque GTG A B 1,000 bp 622 bp 23,130 bp 4,361 bp 2,500 bp 238 bp 242 bp 400 bp Preparation of Agarose Gels Separation of DNA markers in 1% SeaKem GTG and SeaPlaque GTG Agarose gels in 1X TBE Buffer (Prepared from AccuGENE 10X TBE Buffer). Lane A: Lonza's 50-2,500 bp DNA Marker (~0.25 ng/band) Lane B: New England BioLabs Hind III digest of lambda DNA (0.125 mg/lane) 20 cm long gels were run at 6 V/cm for ~ 2.5 hrs. Gels were post stained using Lonza s 1X GelStar Nucleic Acid Gel Stain for 30 minutes. No destain. Resolution Performance of Lonza agaroses for DNA <1 kb Separation of DNA markers in 3% NuSieve 3:1, NuSieve GTG and MetaPhor Agarose gels in 1X TBE (Prepared from AccuGENE 10X TBE Buffer). Lane A: Lonza s 50-1,000 bp DNA Marker (~25 ng/band) Lane B: New England BioLabs Msp I digest of pbr322 (0.125 mg/lane) 20 cm long gels were run at 6 V/cm for 2 hrs. & 20 mins. Gels were post stained using Lonza s 1X GelStar Nucleic Acid Gel Stain for 30 minutes. No destain. 78

87 Section II: Preparation of Agarose Gels Agarose Analytical Specifictions Introduction Lonza performs routine analytical testing on every lot of agarose to ensure lot-to-lot consistency for the critical characteristics that can impact functional performance. Specifications for properties such as gel strength, gelling temperature, electroendosmosis, and others are established at the optimal level for the target applications of the various agarose types. However, analytical specifications alone may not be sufficient for judging the best agarose for a specific application, nor are they the best means to compare agaroses from different vendors. The best way to find out if an agarose is right for your application, is to speak to our Scientific Support representatives, and test a sample of the recommended agarose type(s) based on your application. Lonza has extensive capabilities for modification of agarose properties to fit unique applications and requirements. Ask about our custom capabilities if one our standard products does not meet your needs. Agarose Analytical Specifications Agarose Melting Temperature Gel Strength g/cm 2 Gelling Temperature EEO (-mr) Moisture Sulfate DNA <1 kb NuSieve 3:1 MetaPhor NuSieve GTG 90ºC at 4% 75ºC at 3% 65ºC at 4% ±1,400 at 4% ±300 at 3% ±500 at 4% 32.5ºC 38ºC at 4% 35ºC at 3% 35ºC at 4% % 10% 10% 0.15% NA 0.15% DNA >1 kb PFGE SeaKem LE SeaKem GTG SeaPlaque SeaPlaque GTG SeaKem Gold InCert NA NA 65ºC at 1.5% 65ºC at 1.5% NA 70ºC at 1.5% ±1,200 at 1% ±1,200 at 1% ±200 at 1% ±200 at 1% ±1,800 at 1% ±3,500 at 1.5% ±350 at 1% 36ºC ± 1.5ºC at 1.5% 36ºC ± 1.5ºC at 1.5% 26ºC 30ºC at 1.5% 26ºC 30ºC at 1.5% 34.5ºC 37.5ºC at 1.5% 26ºC 30ºC at 1.5% % 10% 10% 10% 10% 10% 0.15% 0.15% 0.10% 0.10% 0.10% 0.15% Preparation of Agarose Gels Identity Testing I.D.NA NA ±1,300 at 1% 36ºC ± 1.5ºC at 1.5% % 0.15% Protein Electrophoresis SeaKem ME SeaKem HE SeaKem HEEO SeaKem HGT NA NA NA NA ±1,000 at 1% ±650 at 1% ±650 at 1% ±800 at 1% 36ºC ± 1.5ºC at 1.5% 36ºC ± 1.5ºC at 1.5% 36ºC ± 1.5ºC at 1.5% 42ºC ± 1.5ºC at 1.5% ± % 10% 10% 10% 0.20% 0.20% 0.25% 0.30% von Willenbrand s Factor Separation SeaKem HGT(P) NA ±1,000 at 1% 42ºC ± 1.5ºC at 1.5% % 0.20% Isoelectric Focusing IsoGel NA ±500 at 1.5% 35ºC 45ºC Not Detectable 10% 0.20% Cell Culture SeaPrep 50ºC at 1% ±75 at 2% 8ºC 17ºC at 0.8% % 0.10% 79

88 Section II: Preparation of Agarose Gels Suggested Agarose Concentrations and Dye Migration Infomation Table 1: Suggested agaroses for particular applications Table 2: Properties of TAE and TBE Buffer Systems Size Range (Base Pairs) Agarose Type MetaPhor Agarose 50 1,000 NuSieve 3:1 Agarose NuSieve GTG Agarose 1,000 10,000 SeaKem GTG Agarose SeaPlaque GTG Agarose 10,000 SeaKem Gold Agarose Application High resolution analysis; 2% size differences Analysis and blotting; 4% 6% size differences resolved Analysis and blotting; In-gel; 6% size differences resolved Analysis and blotting; recovery required In-gel Analysis Buffer TAE Buffer TBE Buffer Suggested Uses and Comments Use when DNA is to be recovered Use for electrophoresis of large (>12 kb) DNA Low ionic strength Low buffering capacity recirculation may be necessary for extended electrophoretic times Use for electrophoresis of small (<1 kb) DNA Decreased DNA mobility High ionic strength High buffering capacity no recirculation required for extended run times Table 4: Migration of double-stranded DNA in relation to Bromophenol Blue (BPB) and Xylene Cyanol (XC) in agarose gels Preparation of Agarose Gels 80 Table 3: Suggested agarose concentrations for DNA sizes Final Agarose Concentration % (w/v) Size Range (Base Pairs) 1X TAE Buffer 1X TBE Buffer SeaKem LE and SeaKem GTG Agarose 1,000 23, , , , , , NuSieve 3:1 Agarose 500 1, MetaPhor Agarose < SeaPlaque and SeaPlaque GTG Agarose , , , , , , NuSieve GTG Agarose 500 1, SeaKem Gold Agarose 5,000 50, ,000 20, , , TBE Buffer is not recommended for separation of DNA >12,000 bp. 1X TAE Buffer 1X TBE Buffer XC BPB % Agarose XC BPB SeaKem LE and SeaKem GTG Agarose 24,800 2, ,400 2,850 11,000 1, ,000 1,350 10,200 1, , , , , , , , , , , NuSieve 3:1 Agarose MetaPhor Agarose SeaPlaque and SeaPlaque GTG Agarose 11,700 1, , , , , , , , , NuSieve GTG Agarose < < < <20 85 < <20 SeaKem Gold Agarose 24,800 3, ,000 2,550 12,200 2, ,200 1,500 9,200 1, , , , , , , , , , , , Preparation of Agarose Gels

89 Section II: Preparation of Agarose Gels Buffers for Electrophoresis Introduction During electrophoresis, water is electrolyzed, which generates protons at the anode, and hydroxyl ions at the cathode. The cathodal end of the electrophoresis chamber then becomes basic and the anodal end acidic. The use of a buffering system is therefore required when charged molecules are electrophoresed through a separation medium. The two buffers commonly used for DNA electrophoresis are Tris-acetate with EDTA (TAE; 40 mm Tris-acetate, 1 mm EDTA) and Tris-borate with EDTA (TBE; 89 mm Tris-borate, 2 mm EDTA). Because the ph of these buffers is neutral, the phosphate backbone of DNA has a net negative charge and migrates towards the anode. Properties of TAE and TBE Buffer Systems Despite the apparent similarity of TAE and TBE Buffers, each has different properties which make it best suited for different applications (see table below). Buffer TAE Suggested Uses and Properties Use when DNA is to be recovered Use for electrophoresis of large (>12 kb) DNA Low buffering capacity recirculation may be necessary for extended electrophoretic times (>6 hours) DNA Electrophoresis in TAE or TBE Buffer When DNA will not be recovered, either 1X TAE or TBE (1X or 0.5X) Buffer is suitable for use when the DNA is less than 12 kb to 15 kb. For larger DNA, the best buffer to use for electrophoresis is TAE in combination with a low field strength (1-2 V/cm). During these extended electrophoretic runs, larger apparent gel porosity, lower EEO and low field strength decrease the tendency of large DNA to smear. TBE Buffer is preferred for separation of small DNA (<1 kb) when DNA recovery is not required. TBE Buffer s interaction with agarose results in a smaller apparent pore size. The tighter gel reduces the broadening of DNA bands due to dispersion and diffusion. 1X TAE Buffer 1X TBE Buffer % kb 3 kb 1 kb 0.5 kb 12 kb 2 kb 1 kb 0.5 kb Separation of DNA markers in 0.75% to 1.25% SeaKem GTG Agarose gels in 1X TAE and TBE Buffers. 1 kb DNA ladder (Invitrogen, Inc.); 1 μg/lane. The gels were cast in a 25.5 cm framing gel of 1% SeaKem GTG Agarose in a submarine chamber and run under 5 mm of buffer overlay at 5 V/cm for 3 hours, 30 minutes (TBE Buffer), 3 hours (TAE Buffer). Preparation of Agarose Gels TBE Use for electrophoresis of small (<1 kb) DNA Increased resolution of small (<1 kb) DNA Decreased DNA mobility High buffering capacity no recirculation required for extended run times 2% A B 1X TAE Buffer 3% A B 4% A B 2% A B 1X TBE Buffer 3% A B 4% A B Lonza offers ready-to-go buffers for electrophoresis. Refer to AccuGENE Buffer products on page 41. 1,000 bp 1,353 bp 603 bp 100 bp Separation of DNA fragments in 2% to 4% NuSieve 3:1 Agarose gel in 1X TAE and TBE Buffers. Lane A: Lonza s 100 bp Extended Range DNA Ladder (4-7.5 ng/band) Lane B: Invitrogen, Inc.'s Hae III digest of X174 (0.25 ng/band) 20 cm long gels were run at 6 V/cm for 2.5 hrs. in 1X TBE (Prepared from AccuGENE 10X TBE Buffer). Gels were post stained using Lonza s 1X GelStar Nucleic Acid Gel Stain for 30 minutes. No destain. 81

90 Section II: Preparation of Agarose Gels Buffers for Electrophoresis continued Preparation of Agarose Gels Buffer Depth Whichever buffer is used, the depth over the gel in a horizontal electrophoretic system should be 3 mm - 5 mm. Excessive buffer depth will decrease DNA mobility, promote band distortion and can cause excessive heating within the system. Less buffer and the gel may dry out during electrophoresis. The photograph below depicts the effect of buffer depth on DNA electrophoresis. The DNA mobility in the gel with a 10 mm buffer overlay is slower than the gel with a 3 mm buffer overlay. In the Hae III digested X174 marker, the 281/271 bands are starting to resolve in the gel with the 3 mm buffer overlay whereas in the gel with the 10 mm buffer overlay they are not. 1,353 bp 700 bp 400 bp 281 bp 271 bp 100 bp 50 bp Buffer Depletion 10 mm Buffer 3 mm Buffer Overlay Overlay A B A B 1,353 bp 700 bp 400 bp 281 bp 271 bp 234 bp 100 bp 50 bp Separation of DNA markers in a 3% NuSieve 3:1 Agarose gel prepared and run in 1X TBE Buffer. Lane A: DNA marker 50 bp - 1,000 bp DNA Marker (Lonza); 0.4 μg/lane. Lane B: Hae III digested X174 DNA (New England Biolabs); 0.5 μg/lane. The gels were run at 5 V/cm until the bromophenol blue tracking dye was 1 cm from the bottom of each gel. The rate of buffer depletion is in uenced by the buffer used and its buffering capacity. Evidence of buffer depletion is gel melting, smearing of DNA and/or overheating. A 0.5X TBE Buffer has greater buffering capacity than a 1X TAE Buffer at the ph used because the pk a of borate is closer to the initial Buffer ph than that of acetate. Standardsized electrophoresis chambers (15 cm x 30 cm) with a 1.5 L to 2 L capacity will tolerate 40 to 50 Watt hours before buffer depletion, and buffer depletion will not occur in mini-electrophoresis chambers for 10 to 13 Watt hours. Consult the electrophoresis chamber manufacturer for specific values. Effects of buffer depletion and development of a ph gradient can be reduced by recirculating the buffer. This is usually necessary only when electrophoresis is done for extended times or the electrophoresis buffer has a low buffering capacity. Buffer Preparation Alternatively, Lonza offers AccuGENE TBE Buffer in 5X and 10X stock solutions and AccuGENE TAE Buffer in 10X and 50X stock solutions. 50X TAE (Tris-acetate) stock ( 1X=40 mm Tris base, 40 mm acetic acid, 1 mm EDTA) g Tris base 57.1 ml Glacial acetic acid g Na 2 2 O To 1 liter with distilled water Other Buffering Systems Tris-phosphate Buffer (TPE) may also be used for DNA electrophoresis. Like TBE Buffer, TPE has a high buffering capacity, and will not interfere with DNA recovery procedures. However, TPE can not be used when recovered DNA will be used in a phosphate-sensitive reaction. 10X Tris-phosphate Stock (TPE) 5X TBE (Tris-borate) stock (1X=89 mm Tris base, 89 mm boric acid, 2 mm EDTA) (0.5X=45 mm Tris-borate, 1 mm EDTA) 54.0 g Tris base 27.5 g Boric acid 3.72 g Na 2 2 O To 1 liter with distilled water ( 1X=90 mm Tris base, 90 mm phosphoric acid, 2 mm EDTA) g Tris base 15.5 ml 85% Phosphoric acid 7.44 g Na 2 2 O To 1 liter with distilled water Alkaline electrophoresis buffer is used for the analysis of single-stranded DNA. 82

91 Section II: Preparation of Agarose Gels Dissolving Agarose Preparation of Agarose Gels Introduction Agarose undergoes a series of steps when it is dissolved; dispersion, hydration and melting/dissolution. Dispersion Dispersion simply refers to the separation of the particles by the buffer without clumping. Clumping occurs when the agarose starts to dissolve before it is completely dispersed, coating itself with a gelatinous layer which inhibits the penetration of water and keeps the powder inside from dispersing. Dissolution then becomes a long process. Hydration Hydration is the surrounding of agarose particles by a solution (e.g., water or running buffer). Problems are sometimes encountered with hydration when using a microwave oven to dissolve agarose. In part, this occurs because hydration is time dependent and microwave ovens bring the temperature up rapidly. The problem is exacerbated by the fact that the agarose is not being agitated to help dilute the highly concentrated solution around each particle and dissolution is slowed. Melting and Dissolution The final stage in dissolving the agarose is the melting and dissolution. Melting can be done in either a microwave oven or on a hot plate. As the particles hydrate, they become small, highly concentrated gels. Since the melting temperature of a standard agarose gel is about 93 C, merely heating a mixture to 90 C will not completely dissolve agarose. Even low melting temperature agaroses should be boiled to ensure that all the agarose is fully dissolved. Microwave instructions for agarose preparation for gel concentrations of 2% w/v Microwave Instructions for Gel Concentrations <2%w/v 1. Choose a beaker that is 2-4 times the volume of the solution. 2. Add room temperature 1X or 0.5X buffer and a stir bar to the beaker. 3. Sprinkle in the premeasured agarose powder while the solution is rapidly stirred. 4. Remove the stir bar if not Teflon coated. 5. Weigh the beaker and solution before heating. 6. Cover the beaker with plastic wrap. 7. Pierce a small hole in the plastic wrap for ventilation. 8. Heat the beaker in the microwave oven on HIGH power until bubbles appear. Caution: Any Microwave solution may become superheated and foam over when agitated. 9. Remove the beaker from the microwave oven. 10. GENTLY swirl the beaker to resuspend any settled powder and gel pieces. 11. Reheat the beaker on HIGH power until the solution comes to a boil. 12. Hold at boiling point for 1 minute or until all of the particles are dissolved. 13. Remove the beaker from the microwave oven. Caution: Use oven mits when removing beaker from microwave, as container will be hot and may cause burns. 14. GENTLY swirl the beaker to mix the agarose solution thoroughly. 15. After dissolution, add sufficient hot distilled water to obtain the initial weight. 16. Mix thoroughly. 17. Cool the solution to 60 C prior to casting. Materials Microwave oven or hot plate Beaker that is 2-4 times the volume of the solution Te on -coated magnetic stir bar Magnetic stir plate Plastic wrap Oven mitts or other heat protection for hands Reagents Distilled water 1X TAE, 1X TBE or 0.5X TBE Electrophoresis Buffer Agarose powder Caution: Always wear eye protection, and guard yourself and others against scalding solutions. Preparation of Agarose Gels 83

92 Section II: Preparation of Agarose Gels Dissolving Agarose continued Preparation of Agarose Gels 84 Microwave instructions For agarose preparation for gel concentrations 2% w/v 1. Choose a beaker that is 2-4 times the volume of the solution. 2. Add room temperature or chilled buffer (for MetaPhor and NuSieve GTG Agarose) and a stir bar to the beaker. 3. Sprinkle in the premeasured agarose powder while the solution is rapidly stirred to prevent the formation of clumps. 4. Remove the stir bar if not Teflon coated. 5. Soak the agarose in the buffer for 15 minutes before heating. This reduces the tendency of the agarose solution to foam during heating. 6. Weigh the beaker and solution before heating. 7. Cover the beaker with plastic wrap. 8. Pierce a small hole in the plastic wrap for ventilation. For agarose concentrations >4%, the following additional steps will further help prevent the agarose solution from foaming during melting/dissolution: 8a. Heat the beaker in the microwave oven on MEDIUM power for 1 minute. 8b. Remove the solution from the microwave. 8c. Allow the solution to sit on the bench for 15 minutes. 9. Heat the beaker in the microwave oven on MEDIUM power for 2 minutes. Caution: Any microwaved solution may become superheated and foam over when agitated. 10. Remove the beaker from the microwave oven. Caution: Use oven mits when removing beaker from microwave, as container will be hot and may cause burns. 11. GENTLY swirl to resuspend any settled powder and gel pieces. 12. Reheat the beaker on HIGH power for 1-2 minutes or until the solution comes to a boil. 13. Hold at the boiling point for 1 minute or until all of the particles are dissolved. 14. Remove the beaker from the microwave oven. 15. GENTLY swirl to mix the agarose solution thoroughly. 16. After dissolution, add sufficient hot distilled water to obtain the initial weight. 17. Mix thoroughly. 18. Cool the solution to 60 C prior to gel casting. Hot plate instructions for preparing agarose 1. Choose a beaker that is 2-4 times the volume of the solution. 2. Add room temperature or chilled buffer (for MetaPhor or NuSieve GTG Agarose) and a stir bar to the beaker. 3. Sprinkle in the premeasured agarose powder while the solution is rapidly stirred to prevent the formation of clumps. 4. Weigh the beaker and solution before heating. 5. Cover the beaker with plastic wrap. 6. Pierce a small hole in the plastic wrap for ventilation. 7. Bring the solution to a boil while stirring. 8. Maintain gentle boiling until the agarose is dissolved (approximately 5-10 minutes). 9. Add sufficient hot distilled water to obtain the initial weight. 10. Mix thoroughly. 11. Cool the solution to 60 C prior to casting. Materials Microwave oven or hot plate Beaker that is 2-4 times the volume of the solution Te on -coated Magnetic Stir Bar Magnetic stir plate Plastic wrap Reagents Distilled water 1X TAE, 1X TBE or 0.5X TBE Electrophoresis Buffer Agarose powder Caution: Always wear eye protection, and guard yourself and others against scalding solutions.

93 Section II: Preparation of Agarose Gels Casting Agarose Gels Introduction Horizontal gel casting instructions For optimal resolution, cast horizontal gels 3 mm - 4 mm thick (see figure below). The volume of gel solution needed can be estimated by measuring the surface area of the casting chamber, then multiplying by the gel thickness. Thinner gels can be cast on GelBond Film and/ or in a vertical electrophoresis apparatus. The photographs below depict the effect of gel depth on DNA electrophoresis. Gel thickness has a profound effect on the resolution of smaller fragments. The smaller DNA fragments in the 10 mm thick gel are fuzzy, whereas in the 3 mm thick gel the resolution is sharp throughout the gel. There is also a higher background staining in gels thicker than 5 mm. 23,130 bp 4,361 bp 2,027 bp 1,078 bp 310 bp 10 mm Thick Gel A B A 3 mm Thick Gel A B A 23,130 bp 4,361 bp 2,027 bp 1,078 bp 310 bp Separation of DNA markers in 1% SeaKem GTG Agarose gels prepared and run in 1X TBE Buffer. Lane A: Hind III digest of lambda DNA (Boehringer Mannheim); 0.1 μg/lane. Lane B: Hae III digested X174 DNA (New England Biolabs); 0.5 μg/ lane. 20 cm long gels were run at 6 V/cm for 2 hours 15 minutes (10 mm thick gel) and 2 hours 10 minutes (3 mm thick gel). Lonza offers a wide range of precast gels. Refer to page 17 for information. 1. Allow the agarose solution to cool to 60 C. 1. While the agarose solution is cooling: 2a. Assemble the gel casting tray. 2b. Level the casting tray prior to pouring the agarose solution. 2c. Check the comb(s)* teeth for residual dried agarose. Dried agarose can be removed by scrubbing the comb teeth with a lint-free tissue soaked in hot distilled water. 2d. Allow a small space (approximately 0.5 mm - 1 mm) between the bottom of the comb teeth and the casting tray. 3. Pour the agarose solution into the gel tray. 4. Replace the comb(s). 5. Allow the agarose to gel at room temperature for 30 minutes. 6. Low melting temperature agaroses and MetaPhor Agarose require an additional 30 minutes of gelling at 4 C to obtain the best gel handling. The additional cooling step is essential for obtaining fine resolution in MetaPhor Agarose. 7. Once the gel is set, flood with running buffer. 8. Slowly remove the comb. 9. Place the gel casting tray into the electrophoresis chamber. 10. Fill the chamber with running buffer until the buffer reaches 3 mm - 5 mm over the surface of the gel. 11. Gently flush the wells out with electrophoresis buffer using a Pasteur pipette to remove loose gel fragments prior to loading the samples. 12. Load DNA and electrophorese. * The thickness of the comb in the direction of the electric field can affect the resolution. A thin comb ( 1 mm) will result in sharper DNA bands. With a thicker comb, more volume can be added to the well but the separated DNA bands may be broader. Materials Horizontal electrophoresis apparatus Combs Pasteur pipette Reagents Agarose solution Electrophoresis buffer 85 Preparation of Agarose Gels

94 Section II: Preparation of Agarose Gels Casting Agarose Gels continued Preparation of Agarose Gels Vertical Gel Casting Instructions Follow the steps below to cast a vertical agarose gel. This protocol is divided into the following segments: Cassette assembly Cassette sealing Casting the gel Preparing for electrophoresis Cassette assembly Unlike polyacrylamide gels, agarose gels do not adhere to glass plates and may slide out during electrophoresis. To prevent this from happening, frosted glass plates or plastic plates can be used. Follow the steps below to assemble the glass plates. 1. Use clean glass plates. Clean with soap and water, rinse with distilled water and dry. 2. Wipe the plates with ethanol and a lint-free tissue. 3. Place two side spacers on the back plate. Follow the steps below if using glass plates. 3a. For 1 mm thick standard size gels, cut a strip of Whatman 3MM Chromatography paper (1 mm thick and 5-10 mm wide) long enough to fit between the two spacers.* 3b. Wet with running buffer. 3c. Place at the bottom of the back plate in contact with the spacers on each side (see Figure 1, page 87). 4. Put on the front plate. 5. Clamp the glass plates together. 6. Use the manufacturer s casting apparatus or seal the cassette against leaks with silicone tubing or tape. Materials Vertical electrophoresis apparatus Combs and side spacers Whatman 3MM Chromatography Paper Clamps Silicone tubing or electrical tape Two 60 ml syringes with16-gauge needles Heat gun or 55 C oven Scalpel or razor blade Reagents Agarose solution Electrophoresis buffer * Alternatively, use GelBond Film as a support for the gel, which obviates the need for the use of Whatman 3MM Chromatography paper to hold the gel in the cassette during electrophoresis (see Chapter IX). GelBond Film is put into the casting cassette and the gel attaches to the film during the gelling process. After electrophoresis, the gel may be dried down on the GelBond Film and kept as a permanent record. However, GelBond Film blocks UV light below 300 nm and exhibits background uorescence. To overcome these problems, gels cast on GelBond Film may be photographed inverted (gel side down) on the UV light box. Background uorescence can be screened out by using red, orange (Wratten #22 or #25 gelatin filter) and UV filters (Wratten #2B gelatin filter). 86

95 Section II: Preparation of Agarose Gels Casting Agarose Gels continued Cassette Sealing Follow one of the methods below to seal the cassette prior to casting the gel. Silicone tubing method 1. Use silicone tubing which is the same diameter as the spacer thickness. 2. Cut a piece long enough to extend along the bottom and up both sides of the cassette. 3. Place the tubing across the bottom of the back plate below the blotting paper strip (see Figure 2). 4. Place the top plate over the bottom plate. 5. Clamp the glass plates together at the bottom. 6. Run the tubing up either side of the plates and finish clamping the plates together (see Figure 3). Tape method 1. Place the top plate over the bottom plate. 2. Tape the sides of the cassette with separate pieces of tape. 3. Tape the bottom of the cassette with a separate piece of tape. This way the tape on the bottom can be removed for electrophoresis without disturbing the tape at the sides of the gel (see Figure 4). 4. Clamp the plates together. Casting a Vertical Agarose Gel 1. Prepare agarose solution as described previously. 2. Pre-warm the assembled cassette and a 60 ml syringe for 15 minutes by placing in a 55 C oven or by using a heat gun. 3. Cool the dissolved agarose to 60 C. 4. Pour into a pre-warmed 60 ml syringe fitted with a 16-gauge needle. 5. Wedge the needle tip between the plates in the upper corner of the cassette with the needle opening directed toward the back plate (see Figure 5). 6. Inject the agarose solution at a moderate, steady rate. Keep a constant flow to prevent air bubble formation (see Figure 5). 7. Angle the cassette while pouring so the agarose solution flows down one side spacer, across the bottom and up the other side. 8. Fill until the agarose solution goes just above the glass plates. 9. Insert one end of the comb, then slowly insert the rest of the comb until the teeth are at an even depth. Insert the comb into the agarose to the minimal depth necessary to accommodate your samples (see Figure 6, page 88). Figure 1. Whatman 3MM Chromatography Paper Figure 2. Figure 3. Figure 4. Figure 5. Preparation of Agarose Gels Continued on next page. 87

96 Section II: Preparation of Agarose Gels Casting Agarose Gels continued 10. Place extra clamps on the side of the glass plates to hold the comb in place. 11. Cool the gel at room temperature for 15 minutes. 12. Place the gel at 4 C for 20 minutes. 13. Remove the clamps at the top of the gel. 14. Remove any excess agarose with a scalpel or razor blade. 15. Squirt running buffer in the spaces between the comb and the gel. 16. Slowly and gently lift the comb straight up. Allow air or buffer to enter the well area to release the vacuum which forms between the agarose and the comb. 17. The wells can be further cleaned by flushing with running buffer. 18. The gel can be stored overnight in a humidity chamber or in a sealed bag with a buffer-dampened paper towel. Figure 6. Preparing for electrophoresis Preparation of Agarose Gels 1. Remove the silicone tubing or tape at the bottom of the cassette NOTE: If you have placed Whatman 3MM Chromatography Paper between the plates at the bottom, it is not necessary to remove it as it will not interfere with electrophoresis. 2. Place the cassette into the chamber at an angle to minimize the number of bubbles which can collect in the well area. 3. Rinse out well area with a syringe. NOTE: Since agarose does not adhere well to glass, leave as many clips in place as possible. For some electrophoresis chambers, it is helpful to seal the spacers at the top of the gel with molten agarose. 88

97 Section III: Loading and Running DNA in Agarose Gels Loading and Running DNA in Agarose Gels In This Section DNA Loading 90 Loading Buffers 91 Optimal Voltage and Electrophoretic Times 92 Fast Running Protocols for High Resolution in MetaPhor Agarose Gels 93 References 94 89

98 Section III: Loading and Running DNA in Agarose Gels DNA Loading Loading and Running DNA in Agarose Gels Introduction The amount of DNA to load per well is variable. Most important are the quantities of DNA in the bands of interest. Optimal DNA loading amount The amount of DNA that may be loaded on a gel depends on several factors: Well volume Fragment size: The capacity of the gel drops sharply as the fragment size increases, especially over a few kilobases Distribution of fragment sizes Voltage gradient: Higher voltage gradients are better suited to DNA fragments <1 kb, lower voltages are better suited to fragments >1 kb Detection Method: The least amount of dsdna in a single band that can be reliably detected with ethidium bromide is approximately 10 ng, with GelStar Nucleic Acid Gel Stain is approximately 20 pg and with SYBR Green I Stain is 60 pg Overloaded DNA results in trailing and smearing, a problem that will become more severe as the size of the DNA increases. The photograph below shows the effect of overloaded and underloaded DNA on an agarose gel. Where samples are loaded in excess (0.5 μg/lane), you can see band broadening and smearing of the larger molecular weight fragments. Where samples are underloaded, you lose the small molecular weight fragments. The optimum loading level for the marker used in the photograph is 0.1 μg/ lane ,130 bp 6,557 bp The optimal amount of DNA to load in the well may be calculated by the fraction of the total DNA which is in the band of interest, represented by the following: Fragment of interest (kbp) Size of DNA sample (kbp) NOTE: The most DNA compatible with a clean sharp band is approximately 100 ng. For example: The size of your DNA sample is 48.5 kbp and when run on the gel 8 fragments are separated. Your fragment of interest is 2.3 kbp. Calculation: 2.3 kbp 48.5 kbp X 100 = % DNA in band of interest X 100 = 4.7% DNA in fragment of interest If you load 1 μg of DNA, then 4.7% of the 1 μg of loaded sample will appear in your fragment of interest (47 ng). 2,322 bp 564 bp Separation of DNA markers in a 1% SeaKem GTG Agarose gel prepared and run in 1X TBE Buffer. Hind III digested lambda DNA (Boehringer Mannheim) was loaded from left to right at 0.025, 0.05, 0.1, 0.2 and 0.5 μg/lane. 20 cm long gels were run at 6 V/cm for 2 hours, 5 minutes. 90

99 Section III: Loading and Running DNA in Agarose Gels Loading Buffers Introduction Gel loading buffers serve three purposes in DNA electrophoresis: Increase the density of the sample: This ensures that the DNA will drop evenly into the well Add color to the sample: Simplifies loading Add mobility dyes: The dyes migrate in an electric field towards the anode at predictable rates. This enables one to monitor the electrophoretic process Loading buffers At least five loading buffers are commonly used for agarose gel electro phoresis. They are prepared as six-fold concentrated solutions. If needed, 10X solutions of each buffer can also be prepared. Alkaline loading buffer is used when performing alkaline gel electrophoresis. Ficoll based loading buffers To increase the sharpness of DNA bands, use Ficoll (Type 400) Polymer as a sinking agent instead of glycerol. The use of the lower molecular weight glycerol in the loading buffer allows DNA to stream up the sides of the well before electrophoresis has begun and can result in a U-shaped band. In TBE gels, glycerol also interacts with borate which can alter the local ph. Sample preparation Loading buffer that is too high in ionic strength causes bands to be fuzzy and migrate through the gel at unpredictable rates. Ideally, the DNA sample should be resuspended in the same solution as the running buffer. If this is not possible, use a sample buffer with a lower ionic strength than the running buffer. The photograph below shows the effect of high salt concentrations in loading buffers on DNA resolution. Loading Buffer 6X Recipe Storage Temperature Sucrose Based 40% (w/v) Sucrose 4 C 0.25% Bromophenol Blue 0.25% Xylene cyanol FF Glycerol Based Ficoll Based Alkaline 30% Glycerol in distilled water 4 C 0.25% Bromophenol Blue 0.25% Xylene cyanol FF 15% Ficoll (Type 400) room Polymer in distilled water temperature 0.25% Bromophenol Blue 0.25% Xylene cyanol FF 300 mn NaOH 4 C 6 mm EDTA 18% Ficoll (Type 400) Polymer in distilled water 0.15% Bromocresol Green 0.25% Xylene cyanol FF 23 kb 9.4 kb 4.3 kb 2.33 kb 0.56 kb Loading and Running DNA in Agarose Gels Lonza offers ready-to-use DNA loading buffers. Refer to page 41 for information. Separation of Hind III digested lambda DNA (Invitrogen, Inc.) marker (5 μg/lane) in a 1% SeaKem GTG Agarose gel prepared and run in 1X TAE Buffer. 20 cm long gels were run at 6 V/cm for 2 hours. The sample buffer was mixed with varying amounts of NaCl to obtain different final salt concentrations. Lane 1: 4 M NaCl, Lane 2: 3 M NaCl, Lane 3: 1 M NaCl, Lane 4: 0.5 M NaCl, Lane 5: 0.25 M NaCl and Lanes 7-9: No addition of NaCl. 91

100 Section III: Loading and Running DNA in Agarose Gel Optimal Voltage and Electrophoetic Times Optimal voltage The distance used to determine voltage gradients is the distance between the electrodes, not the gel length. If the voltage is too high, band streaking, especially for DNA 12 kb - 15 kb, may result. When the voltage is too low, the mobility of small ( 1 kb) DNA is reduced and band broadening will occur due to dispersion and diffusion. The photographs below show the effect of voltage on small DNA. The small fragments on the gel run at 1 V/cm show severe band broadening and fuzziness. The gel run under 5 V/cm has sharp bands both in the small fragments and the larger fragments. Buffer also plays a role in band sharpness. The photographs below show the effect of voltage and buffer on large DNA. When large DNA is subjected to very high voltage, smearing occurs. Voltage table The table below provides a quick reference for optimal voltage for DNA electrophoresis. Recommended Voltages and Buffers Related to DNA Size and Application Buffer Size Voltage Recovery Analytical 1 kb 5 V/cm TAE TBE 1 kb to 12 kb 4-10 V/cm TAE TAE/TBE >12 kb 1-2 V/cm TAE TAE NOTE: MetaPhor Agarose can also be run at very high voltages to achieve 1% - 2% resolution. See Fast Running Protocols for High Resolution in MetaPhor Agarose Gels, this chapter. Optimal electrophoretic time The gel should be run until the band of interest has migrated 40% - 60% down the length of the gel (see the Dye Mobility Table). Band broadening resulting from dispersion and diffusion results in a decrease in resolution in the lower third of the gel. Resolution may also be decreased in smaller gels, since longer electrophoretic runs result in greater separation between two fragments. Gel 1 A B Gel 2 A B Gel Gel Loading and Running DNA in Agarose Gels 1,353 kb 310 kb 622 kb 160 kb kb 110 kb 67 kb Separation of DNA markers in 3% NuSieve 3:1 Agarose Gels. Lane A: Hae III digested X174 DNA (New England Biolabs); 0.5 μg/lane. Lane B: Msp I digested pbr322 DNA (New England Biolabs); 0.5 μg/lane. Gel 1: 21 cm long gel run at 1 V/cm in 1X TAE Buffer (recirculating) for 16 hours. Gel 2: 20 cm long gel run at 5 V/cm in 1X TBE Buffer for 2 hours, 10 minutes. Separation of Gensura s 5 kb and 2 kb DNA ladders in 0.5% SeaKem LE Agarose gels. Odd numbered lanes are 5 kb ladders; even numbered lanes are 2 kb ladders. Lane 1: 5 ng/band; Lane 2: 5 ng/band; Lane 3: 20 ng/band; Lane 4: 20 ng/band; Lane 5: 60 ng/band; Lane 6: 60 ng/band. Gel 1: 20 cm long gel run at 8 V/cm in 1X TBE Buffer without recirculation. Gel 2: 21 cm long gel run at 1 V/cm in 1X TAE Buffer with recirculation. The FlashGel System (pages 18-25) enables very fast (5 minute) high voltage separation of DNA fragments 10 bp to 10 kb.

101 Section III: Loading and Running DNA in Agarose Gels Fast Running Protocols for High Resolution in MetaPhor Agarose Gels Introduction The protocols in the following section describe how to increase resolution to a 1% size difference with DNA between 100 bp and 500 bp, and decrease your electrophoretic time to 1.5 hours. A standard horizontal submarine gel apparatus can be used to achieve resolution which is comparable to polyacrylamide gels at 8%. MetaPhor Agarose can also be used to achieve similar resolution in a standard vertical gel electrophoretic system. The photographs below show the resolution achieved in 4% MetaPhor Agarose gels, using the same markers in three different formats. Electrophoretic conditions Vertical Rapid Horizontal Format Format Resolution 1% ( 500 bp) 1% ( 500 bp) Run Time hours 1.5 hours Gel Concentration 3% - 4% 3% - 4% Gel Length 16 cm - 26 cm 20 cm Gel Thickness 1 mm 3 mm Gel Buffer 1X TAE or 1X TBE 1X TAE or 1X TBE Running Buffer 1X TAE or 1X TBE 0.5X TAE or 0.5X TBE TBE Voltage* 17 V/cm 17 V/cm Temperature ambient 15 C** *V/cm is determined by the total voltage divided by the interelectrode distance in cm. **Circulate electrophoresis buffer with a recirculator-chiller water bath. Standard Horizontal Format Vertical Format Rapid Horizontal Format bp 527 bp 160 bp 147 bp 76 bp 34 bp Materials Horizontal electrophoresis chamber to accommodate a 20 cm long gel or vertical electrophoresis chamber Power supply Recirculator-chiller water bath Reagents Electrophoresis buffer (TAE or TBE) MetaPhor Agarose GelStar or SYBR Green Nucleic Acid Gel Stains or ethidium bromide solution Caution: Materials and methods shown here present hazards to the user and the environment. Refer to the safety information on page 210 before beginning these procedures. Fast running protocol for horizontal gels NOTE: This protocol cannot be used with only a peristaltic pump in the cold room; the gel will melt. 1. Prepare a 3-4% MetaPhor Agarose gel in 1X electrophoresis buffer. 2. Cast a 3 mm thick, 20 cm long agarose gel. 3. Allow the gel to solidify at room temperature. 4. Place at 4 C for 30 minutes. 5. Place the gel in the electrophoresis chamber. 6. Use 0.5X TBE or 1X TAE Running Buffer in the electrophoresis chamber. 7. Add the running buffer to a depth of 3 mm over the surface of the gel. 8. Load 20 ng - 50 ng of DNA. If this cannot be estimated, load varying amounts of the sample. The sharpest bands are seen with small (5 μl - 10 μl) loading volumes. 9. Run the gel at 17 V/cm (interelectrode distance). 10. When the sample has left the well and moved into the gel, begin recirculating the electrophoresis buffer. 11. Chill and circulate the electrophoresis buffer with a recirculator-chiller water bath. 12. Run the gel for approximately 1.5 hours. Loading and Running DNA in Agarose Gels 4 hours 1.5 hours 1.5 hours Lane 1: 4 bp linker ladder; Lane 2: 8 bp linker ladder; Lane 3: 12 bp linker ladder; Lane 4: 600 ng of 100 bp ladder; Lane 5: 400 ng of pbr322/msp I digest ng of 100 bp ladder; Lane 6: 400 ng of pbr322/msp I digest. 93

102 Section III: Loading and Running DNA in Agarose Gels Fast Running Protocols for High Resolution in MetaPhor Agarose Gels continued Troubleshooting The fast running protocol will not work if the buffer is not chilled or recirculated. The photograph to the right shows the effect of DNA resolution on running the gel in a cold room (4 C), without buffer recirculation. 622 bp Fast running protocol for vertical gels 1. Cast a vertical agarose gel in 1X electrophoresis buffer following the Vertical Gel Casting Instructions on page Carefully flush the wells with running buffer. 3. Load 20 ng - 50 ng of DNA in the band of interest. If this cannot be estimated, load varying amounts of the sample. The sharpest bands are seen with a small (5 μl - 10 μl) loading volume. 4. Run the gel at 17 V/cm interelectrode distance. 5. Run the gel approximately hours. 100 bp Msp I digest of pbr322 (New England Biolabs), 400 ng/lane and Lonza's 100 bp DNA Ladder, 600 ng/lane run on a 4% MetaPhor Agarose gel prepared in 1X TAE Buffer. The gel was run at 17 V/cm in 0.5X TAE Buffer for 90 minutes in the cold room without buffer recirculation. NOTE: The gel melted and was allowed to resolidify prior to staining. Loading and Running DNA in Agarose Gels References 94 FMC BioProducts, Resolutions 9(1), Rickwood, et al., Nucleic Acid Electrophoresis: A Practical Approach, 2nd edition, Oxford University Press, Sambrook J., et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Vandenplas S., et al., J. Medical Genetics 21: , 1984.

103 Section IV: Detection and Sizing of DNA in Agarose Gels In This Section Guide to Lonza Ladders and Markers 96 Detecting DNA with GelStar and SYBR Green Nucleic Acid Gel Stains 98 Detecting DNA with Ethidium Bromide 102 High Sensitivity Detection using the FlashGel System 103 References Detection and Sizing of DNA in Agarose Gels

104 Section IV: Detection and Sizing of DNA in Agarose Gels Guide to Lonza Ladders and Markers Size Range (Bold numbers indicate brighter bands) Detection and Sizing of DNA in Agarose Gels 20 bp 20 bp Ext 100 bp 100 bp ext Tandem 500 bp Quant Ladder Rev Quant Ladder 50 bp bp 50 bp bp Standard Ladders (Cat. No.) NA SimplyLoad Ladders (Cat. No.) NA NA NA Number of Fragments Size Range 20 bp 500 bp 20 bp 1,000 bp 100 bp 1,000 bp 10 bp 3,000 bp 100 bp 12 kb 500 bp 8 kb 100 bp 1,000 bp 100 bp 1,000 bp 50 bp 1,000 bp 50 bp 2,500 bp 1 kb 10 kb 500 bp 1,000 bp 1,000 bp 3,000 bp 12 kb 8,000 bp 1,000 bp 1,000 bp 1,000 bp 2.5 kb 10 kb 480 bp 980 bp 900 bp 2,900 bp 11 kb 7,500 bp 700 bp 700 bp 700 bp 2 kb 7 kb 460 bp 960 bp 800 bp 2,800 bp 10 kb 7,000 bp 500 bp 500 bp 525 bp 1.5 kb 5 kb 440 bp 940 bp 700 bp 2,700 bp 9 kb 6,500 bp 200 bp 200 bp 500 bp 1250 bp 4 kb 420 bp 920 bp 600 bp 2,600 bp 8 kb 6,000 bp 100 bp 100 bp 400 bp 1 kb 3 kb 400 bp 900 bp 500 bp 2,500 bp 7 kb 5,500 bp 300 bp 700 bp 2.5 kb 380 bp 880 bp 400 bp 2,400 bp 6 kb 5,000 bp 200 bp 525 bp 2 kb 360 bp 860 bp 300 bp 2,300 bp 5 kb 4,500 bp 100 bp 500 bp 1.5 kb 340 bp 840 bp 200 bp 2,200 bp 4 kb 4,000 bp 50 bp 400 bp 1 kb 320 bp 820 bp 100 bp 2,100 bp 3 kb 3,500 bp 300 bp 300 bp 800 bp 2,000 bp 2 kb 3,000 bp 200 bp 280 bp 780 bp 1,900 bp 1 kb 2,500 bp 100 bp 260 bp 760 bp 1,800 bp 900 bp 2,000 bp 50 bp 240 bp 740 bp 1,700 bp 800 bp 1,500 bp 220 bp 720 bp 1,600 bp 700 bp 1,000 bp 200 bp 700 bp 1,500 bp 600 bp 500 bp 180 bp 680 bp 1,400 bp 500 bp 160 bp 660 bp 1,300 bp 400 bp 140 bp 640 bp 1,200 bp 300 bp 120 bp 620 bp 1,100 bp 200 bp 100 bp 600 bp 1,000 bp 100 bp 80 bp 580 bp 900 bp 60 bp 560 bp 800 bp 40 bp 540 bp 700 bp 20 bp 520 bp 600 bp 500 bp 500 bp 480 bp 400 bp 460 bp 300 bp 440 bp 200 bp 420 bp 100 bp 400 bp 380 bp 360 bp 340 bp 320 bp 300 bp 280 bp 260 bp 240 bp 220 bp 200 bp 180 bp 160 bp 140 bp 120 bp 100 bp 80 bp 60 bp 40 bp 20 bp 1 kb- 10 kb 96

105 Section IV: Detection and Sizing of DNA in Agarose Gels Guide to Lonza Ladders and Markers continued FlashGel DNA and RNA Markers Size Range 0.5 Kb kb 0.5 Kb kb 100 bp - 3 kb 100 bp - 4 kb 100 bp kb 50 bp kb Ladder/Marker RNA Marker RNA Marker DNA Marker DNA Marker QuantLadder DNA Marker Cat. No Number of Bands kb 9.0 kb 3,000 bp 4,000 bp 1,500 bp 1,500 bp 6.0 kb 5.0 kb 1,500 bp 2,000 bp 800 bp 800 bp 5.0 kb 3.0 kb 800 bp 1,250 bp 400 bp 500 bp 4.0 kb 1.5 kb 500 bp 800 bp 250 bp 300 bp 3.0 kb 1.0 kb 100 bp 300 bp 100 bp 200 bp 2.5 kb 0.5 kb 200 bp 150 bp 2.0 kb 100 bp 100 bp 1.5 kb 50 bp 1.0 kb 0.5 kb FlashGel System Markers and Ladders Catalog No ,000 bp 2,000 bp 1,250 bp 800 bp 500 bp 300 bp 200 bp 100 bp 1,500 bp (30ng) 800 bp (21ng) 400 bp (15 ng) 250 bp (7.5 ng) 100 bp (3 ng) 1,500 bp 800 bp 500 bp 300 bp 200 bp 150 bp 100 bp 50 bp 3,000 bp 1,500 bp 800 bp 500 bp 300 bp 100 bp 3,000 bp 1,500 bp 800 bp 500 bp 300 bp 9.0 Kb 5.0 Kb 0.3 Kb 1.5 Kb 1.0 Kb 0.5 Kb 9 kb 6 kb 5 kb 4 kb 3 kb 2.5 kb 2 kb 1.5 kb 1 kb 0.5 kb 100 bp FlashGel DNA Marker 100 bp4 kb 1.2%, (Standard) FlashGel DNA Marker 100 bp1.5 kb 1.2%, (Quantitation) FlashGel DNA Marker 50 bp1.5 kb 2.2%, (High Concentration) FlashGel DNA Marker 100 bp3 kb 1.2%, (Double-tier) FlashGel RNA Marker 0.5 kb9 kb 1.2%, & (RNA) Lonza RNA Marker 0.5 bp9 kb 1.2%, (RNA) Detection and Sizing of DNA in Agarose Gels 97

106 Section IV: Detection and Sizing of DNA in Agarose Gels Detecting DNA with GelStar, SYBR Green I or II Nucleic Acid Gel Stains Introduction GelStar and SYBR Green Nucleic Acid Gel Stains are highly sensitive uorescent stains for detecting nucleic acids in agarose gels. Unlike ethidium bromide, these stains uoresce only upon binding to nucleic acids. Background staining is minimal and destaining of gels is not required. GelStar Stain gives high detection sensitivity for doublestranded or single-stranded DNA and RNA. SYBR Green I Stain exhibits a preferential affinity for double-stranded nucleic acids and SYBR Green II Stain is most sensitive for single-stranded nucleic acids. Advantages High sensitivity times more sensitive than ethidium bromide Flexible Add GelStar Stain directly to the agarose solution or post-stain your gel with GelStar or SYBR Green Stains Fast No destaining required The table below compares the detection sensitivities of several commonly used staining methods. Samples detected with SYBR Green Stains were post-stained with the dye. Samples detected with Ethidium Bromide or GelStar Stain were detected by in-gel staining. Detection limits were determined by using DNA samples that were serially diluted and by recording the lowest amount that gave a visible band on photographs. Photographic conditions were varied as needed to use the longest exposure time possible without increasing gel background to an unacceptable level. Stain and Method ssdna dsdna GelStar Stain in gel 25 pg 20 pg Ethidium bromide, no destain 1.25 ng 350 pg Ethidium bromide, destain 350 pg 100 pg SYBR Green I or II Stain 60 pg pg Materials Clear polypropylene container (e.g., Rubbermaid Recycling #5 Plastics) GelStar Photographic Filter (Wratten #9 equivalent) or SYBR Green Photographic Filter (Wratten #15 equivalent) Microcentrifuge UV transilluminator, Dark Reader Transilluminator (Clare Chemical Research, Inc.) or CCD imaging system Reagents Buffer between ph (TAE, TBE or TE) GelStar or SYBR Green I or II Stain stock solution Caution: Materials and methods shown here present hazards to the user and the environment. Refer to the safety information on page 210 before beginning these procedures. The photographs below demonstrate the detection sensitivity of GelStar Nucleic Acid Gel Stain when used in-gel or when gels have been post-stained. GelStar Stain In-Gel Post-Stained Lonza s 500 bp DNA Ladder was separated on 1% SeaKem GTG Agarose gels 20 cm long, 4 mm thick, run in 1X TBE Buffer (Prepared from Lonza s AccuGENE 10X TBE Buffer) at 6 V/cm for 3 hours. GelStar Stain was diluted 1:10,000 and added directly to the agarose or the gel was post stained for 30 minutes in a 1:10,000 dilution of GelStar Stain in buffer. Lane 1: 10 ng DNA/ band; Lane 2: 5 ng DNA/band; Lane 3: 2.5 ng DNA/band; Lane 4: 1.25 ng DNA/band. Detection and Sizing of DNA in Agarose Gels The FlashGel System includes gel cassettes pre-stained with a similar high sensitivity stain. Refer to page

107 Section IV: Detection and Sizing of DNA in Agarose Gels Detecting DNA with GelStar, SYBR Green I or II Nucleic Acid Gel Stains continued Tips for staining gels with GelStar or SYBR Green I Stain Follow the guidelines below to increase the detection sensitivity of GelStar or SYBR Green Stains New clear polypropylene containers (e.g., Rubbermaid Recycling #5 Plastics) should be obtained for use with GelStar and SYBR Green Stains; when stored in the dark in polypropylene containers, the diluted stain can be used for up to 24 hours and will stain 2 to 3 gels with little decrease in sensitivity; the containers should be rinsed with distilled water (do not use detergents) after each use and dedicated to GelStar or SYBR Green Stain use only These stains bind to glass and some non-polypropylene (polystyrene) plastics resulting in reduced or no signal from the nucleic acid A 1X working solution of GelStar or SYBR Green Stain should be prepared just prior to use from the 10,000X stock solution by diluting in a ph 7.5 to 8.5 buffer (e.g., TAE, TBE or TE) Agarose gels should be cast no thicker than 4 mm. As gel thickness increases, diffusion of the stain into the gel is decreased, lowering the efficiency of DNA detection Optimal sensitivity for GelStar and SYBR Green Stains is obtained by using the appropriate photographic filters for each stain GelStar Stain: Wratten or Tiffen #9 Filter SYBR Green Stains: Wratten or Tiffen #15 Filter We do not recommend photographing gels with a 254 nm transilluminator; an outline of the UV light source may appear in photographs; a filter that will allow a 525 nm transmission and exclude infrared light is required Procedure for staining DNA with GelStar or SYBR Green Stains For optimal resolution, sharpest bands and lowest background, stain the gel with GelStar or SYBR Green Stain following electrophoresis. Alternatively, GelStar Stain can be included in the agarose gel. It is not recommended to include SYBR Green Stains in the agarose gel. When the dye is incorporated into the agarose, the gel is more sensitive to DNA overloading, and the electrophoretic separation of DNA may not be identical to that achieved with ethidium bromide. The photograph below demonstrates the effect of adding SYBR Green I Stain to the agarose. In-Gel SYBR Green Gel Stain Post-Stained DNA markers were separated on 1% SeaKem Gold Agarose gels 10-cm-long, 3 mm thick, run in 1X TBE Buffer for hours. SYBR Green I Stain was diluted 1:10,000 and added directly to the agarose or the gel was post stained for 30 minutes in a 1:10,000 dilution of SYBR Green Stain in Buffer. Lanes 1-4: BstE II digest of lambda DNA; 1:2 dilutions with an initial loading of 25 ng/lane. Lane 5: 1 kb DNA ladder (Invitrogen, Inc.); 200 ng/lane (SYBR Green I stained gel); 1 μg/lane (Ethidium bromide stained gel). Lane 6: Lonza s 1-10 kb DNA Ladder; 100 ng/lane. Lane 7: Gensura s 1 kb DNA Ladder; 100 ng/lane. Lane 8: Lonza s 100 bp DNA Ladder; 100 ng/lane. Detection and Sizing of DNA in Agarose Gels 99

108 Section IV: Detection and Sizing of DNA in Agarose Gels Detecting DNA with GelStar, SYBR Green I or II Nucleic Acid Gel Stains continued Detectionand Sizing of DNA in Agarose Gels Follow the steps below to stain DNA after electrophoresis 1. Remove the concentrated stock solution of GelStar or SYBR Green Stain from the freezer and allow the solution to thaw at room temperature. 2. Spin the solution in a microcentrifuge to collect the dye at the bottom of the tube. 3. Dilute the 10,000X concentrate to a 1X working solution (1 μl per 10 ml), in a ph buffer, in a clear plastic polypropylene container. Prepare enough staining solution to just cover the top of the gel. 4. Remove the gel from the electrophoresis chamber. 5. Place the gel in staining solution. 6. Gently agitate the gel at room temperature. 7. Stain the gel for minutes. The optimal staining time depends on the thickness of the gel, concentration of the agarose, and the fragment size to be detected. Longer staining times are required as gel thickness and agarose concentration increase. 8. Remove the gel from the staining solution and view with a 300 nm UV transilluminator, CCD camera or Dark Reader Transilluminator (Clare Chemical Research, Inc.). GelStar and SYBR Green Stained Gels do not require destaining. The dyes fluorescence yield is much greater when bound to DNA than when in solution. Follow this procedure when including GelStar Stain in the agarose gel. 1. Remove the concentrated stock solution of GelStar Stain from the freezer and allow the solution to thaw. 2. Spin the solution in a microcentrifuge tube. 3. Prepare the agarose solution (see pages 83-84). 4. Once the agarose solution has cooled to 70 C, add the stain by diluting the stock 1:10,000 into the gel solution prior to pouring the gel (1 μl per 10 ml). 5. Slowly swirl the solution. 6. Pour the gel into the casting tray (see page 85). 7. Load your DNA onto the gel. 8. Run the gel. 9. Remove the gel from the electrophoresis chamber. 10. View with a 300 nm UV transilluminator, CCD camera or Dark Reader Transilluminator (Clare Chemical Research, Inc.). GelStar Stained gels do not require destaining. The dye s fluorescence yield is much greater when bound to DNA than when in solution. Staining vertical gels with GelStar and SYBR Green Stains Incorporating GelStar and SYBR Green Stains into the gel or prestaining the DNA for use in a vertical format is not recommended. The dye binds to glass or plastic plates and DNA may show little to no signal. Gels should be poststained as described in the previous section. Follow this procedure when staining vertical gels with GelStar or SYBR Green Stain 1. Remove the concentrated stock solution of GelStar or SYBR Green Stain from the freezer and allow the solution to thaw at room temperature. 2. Spin the solution in a microcentrifuge to collect the dye at the bottom of the tube. 3. Dilute the 10,000X concentrate to a 1X working solution, in a ph buffer, in a clear plastic polypropylene container. Prepare enough staining solution to just cover the top of the gel. 4. Remove the gel from the electrophoresis chamber. 5. Open the cassette and leave the gel in place on one plate. 6. Place the plate, gel side up in a staining container. 7. Gently pour the stain over the surface of the gel. 8. Stain the gel for 5-15 minutes. 9. View with a 300 nm UV transilluminator, CCD camera or Dark Reader Transilluminator (Clare Chemical Research, Inc.). GelStar or SYBR Green Stained gels do not require destaining. The dye s fluorescence yield is much greater when bound to DNA than when in solution. 100

109 Section IV: Detection and Sizing of DNA in Agarose Gels Detecting DNA with GelStar, SYBR Green I or II Nucleic Acid Gel Stains continued Visualization by Photography Gels stained with GelStar and SYBR Green Stains exhibit negligible background uorescence, allowing long film exposures when detecting small amounts of DNA. Use the appropriate photographic filter for the stain you are using. The table below provides suggested film types and photographic conditions Polaroid Film f-stop Exposure time Type 57 or seconds Type seconds Visualization by image capture system For the best results and optimal sensitivity, visualize GelStar Stained Gels on The Dark Reader Transilluminator (Clare Chemical Research, Inc.) GelStar and SYBR Green Stains are compatible with most CCD and video imaging systems. Due to variations in the filters for these systems, you may need to purchase a new filter. Lonza does not sell filters for this type of camera. Contact your systems manufacturer and using the excitation and emission information listed, they can guide you to an appropriate filter. Stain Emission (nm) Excitation (nm) GelStar Stain SYBR Green I Stain SYBR Green II Stain Application notes The ffuorescent characteristics of GelStar and SYBR Green Stains make them compatible with argon ion lasers. These stains are removed from double-stranded DNA by standard procedures for ethanol precipitation of nucleic acids. Gels previously stained with ethidium bromide can subsequently be stained with GelStar or SYBR Green Stain following the standard protocol for post-staining. There will be some decrease in sensitivity when compared to a gel stained with only GelStar or SYBR Green Stain. The inclusion of GelStar and SYBR Green Stains in cesium chloride density gradient plasmid preparations is not recommended. The effect of the dye on the buoyant density of DNA is unknown. These stains do not appear to interfere with enzymatic reactions. We recommend the addition of 0.1% to 0.3% SDS in the prehybridization and hybridization solutions when performing Southern blots on gels stained with these dyes. Double-stranded DNA-bound GelStar or SYBR Green Stain ffuoresces green under UV transillumination. Gels that contain DNA with single-stranded regions may ffuoresce orange rather than green. Decontamination Staining solutions should be disposed of by passing through activated charcoal followed by incineration of the charcoal. For absorption on activated charcoal, consult Sambrook, et al., pp , (1989). Follow state and local guidelines for decontamination and disposal of Nucleic Acid staining solutions Detection and Sizing of DNA in Agarose Gels 101

110 Section IV: Detection and Sizing of DNA in Agarose Gels Detecting DNA with Ethidium Bromide Detection and Sizing of DNA in Agarose Gels Introduction Ethidium bromide is a ffuorescent dye which detects both single- and double-stranded DNA. However, the affinity for single-stranded DNA is relatively low compared to double-stranded DNA. Ethidium bromide contains a planar group which intercalates between the bases of DNA and, when bound to DNA, results in an increase in ffuorescence yield. Ethidium bromidestained DNA is detected by ultraviolet radiation. At 254 nm, UV light is absorbed by the DNA and transmitted to the dye; at 302 nm, and 366 nm, UV light is absorbed by the bound dye itself. In both cases, the energy is re-emitted at 590 nm in the red-orange region of the visible spectrum. Procedure For optimal resolution, sharpest bands and lowest background, stain the gel with ethidium bromide following electrophoresis. Ethidium bromide can also be included in the gel and electrophoresis buffer (0.5 μg/ml) with only a minor loss of resolution. The electrophoretic mobility of DNA will be reduced by approximately 15%. Follow the steps below to stain DNA after electrophoresis 1. Prepare enough working solution of ethidium bromide. (0.5-1 μg/ml of ethidium bromide in distilled water or gel buffer) to cover the surface of the agarose gel. 2. Remove the gel from the electrophoresis chamber. 3. Submerge the gel for 20 minutes in the ethidium bromide solution. 4. Remove the gel from the solution. 5. Submerge the gel for 20 minutes in a new container filled with distilled water. 6. Repeat in fresh distilled water. 7. Gels can be viewed with a hand-held or tabletop UV light. For gel concentrations of 4% or greater, these times may need to be doubled. If after destaining the background is still too high, continue to destain. Materials Staining vessel larger than gel UV transilluminator Magnetic stir plate Magnetic stir bar Reagents Ethidium bromide stock solution (10 mg/ml) 1.0 g ethidium bromide 100 ml distilled water Stir on magnetic stirrer for several hours Transfer the solution to a dark bottle Store at room temperature Electrophoresis buffer or distilled water Caution: Materials and methods shown here present hazards to the user and the environment. Refer to the safety information on page 210 before beginning these procedures. Follow the steps below when including ethidium bromide in the agarose gel 1. Prepare agarose solution (see Section II). 2. While the agarose solution is cooling, add ethidium bromide to a final concentration of 0.1 to 0.5 μg/ml to the solution. 3. Gently swirl the solution. 4. Pour the gel into the casting tray. 5. Add ethidium bromide to the running buffer to a final concentration of 0.5 μg/ml. 6. Load and run the gel (see Section III). 7. Destain the gel by submerging the gel in distilled water for 20 minutes. 8. Repeat in fresh distilled water. 9. Gels can be viewed with a hand-held or tabletop UV light during or after electrophoresis. Decontamination of ethidium bromide solutions Decontamination of ethidium bromide solutions is described in Sambrook, et al., pp (1989). Follow local guidelines and regulations for ethidium bromide decontamination and disposal. 102

111 Section IV: Detection and Sizing of DNA in Agarose Gels High Sensitivity Detection using The FlashGel System Introduction The FlashGel System uses a proprietary stain that is 5-20 times more sensitive than ethidium bromide stain. Samples prepared at DNA concentrations one-fifth of the concentration typically required for ethidium bromide stained gels will clearly resolve on FlashGel Cassettes. DNA levels of 5 ng per band or more are visible on the lighted FlashGel Dock under most ambient light conditions. DNA levels as low as 0.1 ng per band can be detected on gel images and photos. DNA levels can be adjusted to provide best performance depending upon the image analysis system used (Figure 1). Figure1: DNA concentration detectable with the FlashGel System Because the system is so sensitive, a load volume of 5 l or less is recommended for best performance. Samples may be diluted with FlashGel Loading Dye (for best results), or with water or other common buffers (e.g., TE Buffer) before adding a loading dye. See the table in Figure 2 for examples of sample and marker dilutions in a FlashGel Cassette compared to an ethidium bromide stained agarose gel. The ethidium stained gel required at least 4 times more sample than the FlashGel Cassette. One-tenth the concentration of the FlashGel DNA Marker and QuantLadder, and one-twentieth the concentration of the PCR products were required for good sensitivity of detection on FlashGel Cassettes compared to ethidium bromide gel. Figure2: FlashGel Cassette vs. Ethidium Bromide Gel kb 500 bp 4 kb FlashGel Cassette kb ng/band kb kb 500 bp 4 kb 500 kb Ethidium Bromide Gel ng/band kb 1.2% FlashGel Cassettes, 12+1 well format. 275 volts for 7 minutes. DNA concentrations are ng/band Lanes 1-12: Sample dilution series of 400 bp & 1,500 bp purified fragments (BioVentures) Lane 13: FlashGel DNA Marker. Photographed on UV Transilluminator or Dark Reader Transilluminator. Dark Reader Transilluminator Gel illuminated on the Dark Reader with Dark Reader orange filter in place. Photographed with CCD imager (EtBr filter in place) at 2 second exposure. UV transilluminator: Gel illuminated on UV transilluminator. Photographed with CCD imager (EtBr filter in place) at 2 second exposure. FlashGel Loading Dye was added to all samples prior to loading. Lanes 1&7: FlashGel DNA Marker; Lanes 2&8: FlashGel QuantLadder; Lanes 3&9: Lonza 100 bp Ladder; Lanes 4&10: Lonza 50-2,500 bp Marker; Lanes 5&11: 285 bp -Actin PCR; Lanes 6&12: 294 bp Ambion control PCR. Samples diluted with 1X FlashGel Loading Dye prior to loading. Dilutions and load volumes optimized for each sample in each gel system. See table below. Lane Nos. Sample Type Load volumes and dilutions FlashGel Cassette Ethidium Bromide Gel Lanes 1 & 7 FlashGel DNA Marker 5 μl 1:5 dilution 10 μl undiluted Lanes 2 & 8 FlashGel QuantLadder 5 μl 1:5 dilution 10 μl undiluted Lanes 3 & 9 Lonza 100 bp Ladder 3 μl 1:15 dilution 12 μl 1:15 dilution Lanes 4 & 10 Lonza bp Marker 3 μl 1:5 dilution 12 μl 1:5 dilution Lanes 5 & bp -Actin PCR 5 μl 1:50 dilution 2 μl undiluted PCR rxn Lanes 6 & bp Control PCR (Ambion) 5 μl 1:50 dilution 2 μl undiluted PCR rxn Lane 13 FlashGel DNA Marker 5 μl 1:5 dilution Detection and Sizing of DNA in Agarose Gels 103

112 Section IV: Detection and Sizing of DNA in Agarose Gels High Sensitivity Detection using The FlashGel System continued Refer to The FlashGel System Protocol for complete user instruction, safety and environmental precautions. Some components and technology of the FlashGel System are sold under licensing agreements. The nucleic acid stain in this product is manufactured and sold under license from Molecular Probes, Inc., and the FlashGel Cassette is sold under license from Invitrogen IP Holdings, Inc, and is for use only in research applications or quality control, and is covered by pending and issued patents. The FlashGel Dock technology contains Clare Chemical Research, Inc. Dark Reader Transilluminator technology and is covered under US Patents 6,198,107; 6,512,236; and 6,914,250. The electrophoresis technology is licensed from Temple University and is covered under US Patent 6,905,585. Detection and Sizing of DNA in Agarose Gels 104 References Carmichael, G.G. and McMaster, G.K. Meth. Enzymol. Academic Press, Inc Flores, N., et al., BioTechniques 13(2): , FMC BioProducts, Resolutions 11(2), McMaster G.K. and Carmichael, G.G., Proc. Natl. Acad. Sci. 74: , Peats, S., Anal. Biochem. 140: , Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Schneeberger, C., et al., PCR Methods & App. 4: , 1995.

113 Section V: In-Gel Reactions In-Gel Reactions In This Section Overview 106 Tips for Increasing the Efficiency of In-Gel Reactions 107 Cloning in the Presence of Agarose 108 Restriction Digestion in the Presence of Agarose 112 DNA Amplification in the Presence of Agarose 113 References

114 Section V: In-Gel Reactions Overview In-Gel Reactions Introduction A variety of enzymatic reactions can be performed in the presence of agarose. In-gel reactions are an alternative approach to standard DNA recovery techniques and provide a multitude of benefits. The use of in-gel reactions will not only save time, but eliminate potential sample loss during DNA recovery from an agarose gel. Advantages Saves time otherwise used for recovery Avoids recovery losses Avoids recovery damage, especially shearing of higher molecular weight DNA Compatible agaroses In-gel reactions require the use of a low melting temperature (65 C) agarose. Lonza has developed two high quality Genetic Technology Grade (GTG ) Agaroses for this application, specifically NuSieve GTG and SeaPlaque GTG Agarose. These agaroses are specifically designed and tested for compatibility with in-gel reactions. NuSieve GTG Agarose is the choice for separation of nucleic acids 1,000 bp with a resolving power of 4% - 6% difference in DNA size SeaPlaque GTG Agarose is the choice for separation of large nucleic acid fragments >1,000 bp Applications Enzymes active in the presence of low melting temperature agaroses include: Alkaline phosphatase BAL 31 nuclease DNA polymerase I Klenow fragment Restriction endonucleases Reverse transcriptases T4 DNA ligase T4 DNA polymerase T4 polynucleotide kinase T7 DNA ligase T7 DNA polymerase (Sequenase Polymerase) Taq DNA polymerase The figures below demonstrate the efficiency of in-gel cloning in our low melting temperature agaroses versus sample recovery and also the consistency between different agarose lots. Efficiency of In-Gel Cloning from SeaPlaque GTG Agarose No. Transformants per g of Vector 8.0x x x x x x x x In-gel Recovered Two lanes of Bst E II digest of lambda DNA were electrophoresed in a 1% SeaPlaque GTG Agarose gel prepared in 1X TAE. The 2.3 kb band was excised from each lane. One sample was ligated directly to puc 19 in the presence of remelted agarose (In-gel) and the other sample was purified using a chaotrope-based recovery protocol (Recovered). Transformation into DH5 Cells was done following standard protocols. A 377 bp fragment was electrophoresed in three separate lots of a 3% NuSieve GTG Agarose gel in 1X TAE Buffer then ligated to M13 in the presence of remelted agarose. Transformation into DH5 F Cells was done in remelted agarose following standard protocols. Results 1, 2 and 3 are from three separate lots of NuSieve GTG Agarose. Efficiency of In-Gel Cloning from NuSieve GTG Agarose No. Transformants per g of Vector 4.0x x x x x x x x Lot 1 Lot 2 Lot 3 106

115 Section V: In-Gel Reactions Tips for Increasing the Efficiency of In-Gel Reactions Electrophoretic tips Use a low EDTA (0.1 mm) 1X TAE Buffer; increases the availability of Mg +, a necessary cofactor in many enzymatic reactions Brie y stain the gel after electrophoresis with GelStar or SYBR Green I Nucleic Acid Gel Stain or ethidium bromide; the presence of ethidium bromide during electrophoresis can result in the degradation of DNA Visualize your DNA with a UV light source that is 300 nm. Short UV wavelength light can damage DNA; alternatively, DNA can be detected using Clare Chemical s Dark Reader Transilluminator Minimize exposure of DNA to UV light to less than 1 minute The addition of 1 mm guanosine or cytidine to the gel and electrophoresis buffer is effective in protecting DNA against UV-induced damage; the nucleosides will not impede electrophoresis, detection or purification of DNA. Limit the amount of DNA in the band of interest to 100 ng if possible; more DNA may cause smearing and a wide band will result in an unacceptably large piece of gel to be excised and melted. Effect of Agarose Concentration and Buffer on In-Gel PCR Amount of Amplification per % of Control H 2 0 TAE TBE Agarose Concentration in Reaction Mixture (%) Reaction tips Melt the gel slice at 65 C - 70 C. Do not exceed 70 C as this may cause melting of your DNA Thoroughly mix the reaction components at 37 C - 40 C this ensures proper mixing of DNA with reaction components Do not exceed 0.5% SeaPlaque GTG or 1.5% NuSieve GTG Agarose in the reaction mix Increase the efficiency of blunt end ligations; the addition of larger amounts of vector, insert DNA and T4 DNA ligase or the addition of 5% (w/v) PEG-8000 to the ligation mixture and incubating the ligation reaction mixture for a longer time, can increase the efficiency of the reaction; an alternative strategy is to add linkers with sticky ends to the insert DNA When transforming cells, dilute the melted ligation reaction mixture five-fold before adding to cells When transforming bacterial cells, dilute the agarose containing the ligated DNA further with warmed (40 C) TAE or sterile distilled water before adding the ligation reaction to the competent cells; it is better to add 10 μl of a five-fold diluted mixture than 2 μl of a more concentrated reaction mixture; by doing this, it is less likely that your ligated DNA will be trapped in the agarose when it is added to the chilled cells; also be sure that you do not add excessive DNA to the competent cells which can decrease the transformation efficiency Effect of UV Irradiation on In-Gel Cloning No. of Recombinants Per g Vector 4.0x x x10 7 In-Gel Reactions A 500 bp PCR product was separated on 4% NuSieve GTG Agarose in either 1X TAE or 1X TBE Buffer. Following electro phoresis, the PCR product was reamplified with the same primers as in the original amplification. Relative efficiency was measured by comparing the products on an agarose gel. 1.0x UV Exposure Time (Minutes) A 2 kb fragment, separated in 1% SeaPlaque GTG Agarose and stained with ethidium bromide, was exposed to 300 nm UV irradiation for different lengths of time before excision from the gel, ligation into pbr322, and bacterial cell transformation. The mean of the number of white colonies per microgram vector on two plates is given. Reference Gründemann, D. and Schömig, E., BioTechniques 21: ,

116 Section V: In-Gel Reactions Cloning in the Presence of Agarose In-Gel Reactions Timetable 1. (Day 1) Preparation of vector. Preparation of insert. Preparation of competent cells. 2. (Day 2) Ligation reaction. Transformation reaction. 3. (Day 3) Assess the results obtained from the ligationtransformation reaction. Tips Use 1X TAE Buffer with 0.1 mm EDTA. Electrophorese the DNA without ethidium bromide in the gel. Minimize the exposure time of the DNA to less than 1 minute under UV light. Do not exceed a final concentration of 0.5% SeaPlaque GTG or 1.5% NuSieve GTG Agarose in the reaction mixture. Dilute the agarose solution when running in-gel transformations. Do not exceed >0.02% agarose in the transformation reaction. NOTE: Electroporation can not be used with in-gel ligation/ transformation procedures. Effect of Agarose Concentration on Transformation No. of Recombinants Per g Vector 4.0x x x x Agarose Concentration (%) Aliquots of agarose solutions (0.25%, 0.5%, 1.0% and 2.0% in 1X TAE) were added to puc19 monomer DNA which had been placed in labeled tubes. Aliquots (6 μl=0.01 ng DNA) of the samples containing agarose were added to 100 μl of (Invitrogen, Inc.) frozen competent cells on ice. The agarose concentrations shown are those in the tubes of competent cells after samples were added. Transformations were carried out following standard protocol. Points shown on the graph represent the mean of three transformations. Materials Sterile microcentrifuge ( 1 ml) and polypropylene tubes (17 mm x 100 mm) Horizontal electrophoresis chamber Scalpel or razor blade Heating block or water bath Ice bucket and ice Reagents SeaPlaque GTG or NuSieve GTG Agarose GelStar or SYBR Green I Nucleic Acid Gel Stain or ethidium bromide Distilled water T4 DNA ligase Competent cells Restriction enzymes Calf intestine alkaline phosphatase 1X TAE (0.1 mm EDTA) Gel and Running Buffer Reagents for phenol extraction (Section VI) 10 mm Tris-HCl, ph 7.5 Ethanol 10X T4 ligation buffer 500 mm Tris-HCl, ph 7.5; 100 mm MgCl ; 50 mm Dithiothreitol; 10 mm ATP ph 7.6; μg/μl nuclease-free BSA (bovine serum albumin) Caution: Materials and methods shown here present hazards to the user and the environment. Refer to the safety information on page 210 before beginning these procedures. 108

117 Section V: In-Gel Reactions Cloning in the Presence of Agarose continued Preparing the vector 1. Cut vector with appropriate restriction enzymes. 2. Dephosphorylate vector with calf intestine alkaline phosphatase. 3. Phenol extract vector. 4. Ethanol precipitate vector. Preparing the insert 1. Cut DNA to be used as insert with appropriate restriction enzymes. 2. Electrophorese DNA (100 ng) in a low melting temperature agarose gel prepared in 1X TAE Buffer (0.1 mm EDTA). 3. Briefly stain DNA with GelStar or SYBR Green I Nucleic Acid Gel Stain or ethidium bromide. 4. Excise gel band containing insert and place in a sterile, preweighed tube. 5. Estimate the volume of the gel slice based upon the weight of the slice (e.g., 100 mg = 100 μl). 6. Determine the concentration of the DNA in the gel slice by assuming 100% recovery of DNA in the slice. 7. Store excised band at 4 C until ready for use. 8. Prepare agarose plates with the appropriate selective media for the next day s use. Ligation reaction 1. Remelt the agarose slice by heating to 68 C for 10 minutes. 2. Place the remelted gel slice at 37 C until needed. 3. For a final reaction mixture of 50 μl, add the following components in the following order: Volume Component 18 μl 10 mm Tris-HCl, ph 7.5 or distilled H20 5 μl 10X T4 DNA ligation buffer 1 μl Vector (amount sufficient to obtain a molar ratio of insert to vector of 3-4:1) 1 μl T4 DNA ligase (between 1-2 units) 4. Mix the components gently with a pipette. 5. Add up to 25 μl of the remelted agarose gel slice [25 ng of insert DNA]. Do not exceed a final concentration of 0.5% SeaPlaque GTG or 1.5% NuSieve GTG Agarose in the reaction mixture. 6. Mix components by resuspending with a pipette. 7. Ligate at room temperature for 2-3 hours. In-Gel Reactions Preparing competent cells If you prepare your own competent cells, set up a small overnight culture of cells. High transformation efficiencies can be obtained with frozen competent cells that are either prepared in the laboratory or purchased. Similar transformation efficiencies of 10 7 transformants/μg puc18 DNA with insert have been obtained using the Hanahan Procedure (see citation at end of section); transformation efficiencies are reduced when using the CaCl 2 method. NOTE: Electroporation can NOT be used with in-gel ligation/transformation procedures. 109

118 Section V: In-Gel Reactions Cloning in the Presence of Agarose continued In-Gel Reactions Transformation reaction The transformation procedure outlined below is a modification of the Invitrogen procedure included with their DH5 Competent Cells. When using other frozen competent cells follow manufacturer s directions. 1. Prepare competent cells if working with fresh cells. If using frozen competent cells, remove cells from storage and thaw on ice. 2. Heat the ligation reaction at 68 C for 5 minutes to remelt the agarose. 3. Add competent cells into a prechilled polypropylene tube (17 mm x 100 mm). We use 100 μl of thawed DH5 cells. 4. Add 1 μl - 2 μl of diluted ligation reaction (70 pg pg vector) to competent cells. 5. Mix and incubate on ice for 30 minutes. NOTE: Exceeding 0.02% SeaPlaque GTG or NuSieve GTG Agarose concentration in the final mixture may reduce transformation efficiencies. 6. Heat shock the cells by placing the tubes in a 42 C water bath for 45 seconds. 7. Place the cells back on ice for 2 minutes. 8. Add 0.9 ml of SOC medium to cells. 9. Incubate at 37 C with shaking for minutes. 10. Spread 1 μl μl of the transformation mixture onto an agar plate with the appropriate selective medium. 11. Allow the liquid to soak in. 12. Invert the plates. 13. Incubate overnight at 37 C. Reagents SOC medium 2.0 g Bacto-tryptone, 0.5 g Yeast extract, 1.0 ml 1 M NaCl 0.25 ml 1 M KCl Dissolve in a final volume of 100 ml distilled water; autoclave Add aseptically: 1.0 ml 2 M Mg2+ (1 M MgCl 2, 1 M MgSO 4, filter sterilized) 1.0 ml 2 M glucose (filter sterilized) LB Agar Plates Supplemented with Ampicillin and X-gal (1 L) 10.0 g tryptone 5.0 g yeast extract 10.0 g NaCl 15.0 g agar 800 ml distilled water Stir to dissolve components Adjust to ph 7.5 with NaOH Adjust volume to 1 L with distilled water Carefully heat while stirring until agar is dissolved Remove stir bar and sterilize the medium by autoclaving 25 minutes Place the ask of medium in a water bath set at 50 C and let the medium cool for at least 30 minutes While medium is cooling, prepare ampicillin and X-gal stock solutions. Add 1 ml of ampicillin stock and 2 ml of X-gal stock to the tempered medium and pour into plates Allow plates to sit on bench overnight to dry; then package and store at 4 C Ampicillin stock mg of ampicillin to 1 ml of sterile distilled water X-gal (5-bromo-4-chloro-3-indoyl-ß-D-galactoside) stock 40.0 mg of X-gal to 2 ml of dimethylformamide 110

119 Section V: In-Gel Reactions Cloning in the Presence of Agarose continued Controls We recommend the following controls be run in parallel with the test plates: Vector Ligase # of blue # of white Reason for control colonies colonies puc 18-native Cell competency test puc18-singly digested Background for ligation efficiency efficiency puc18-singly digested Determines ligation efficiency puc18-doubly digested Unrestricted vector background background puc18-doubly digested Determines the efficiency of double digestion puc18-native Reagent check In-Gel Reactions Assessing the results Count colonies and assess the results of ligationtransformation. Colonies containing recombinant plasmids should be white. Blue colonies result from non-recombinant puc18 plasmid. References Hanahan, D., J. Mol. Biol. 166: , Crouse, G.F., et al., Meth. Enzymol. 1: 78-89, Frischauf, A.M., et al., Nucl. Acids Res. 8: , Majumdar, D., et al., BioTechniques 7: , Murray, J.A.H., Nucl. Acids Res. 14: 10118, Struhl, K., BioTechniques 3: ,

120 Section V: In-Gel Reactions Restriction Digestion in the Presence of Agarose In-Gel Reactions Procedure NOTE: The urea sample buffer prevents the samples from regelling after digestion. It will also result in good separation of the DNA during electrophoresis. 1. Electrophorese DNA (several μg) in a low melting temperature agarose prepared in 1X TAE Buffer (0.1 mm EDTA). 2. Briefly stain the gel with GelStar or SYBR Green I Nucleic Acid Gel Stain or ethidium bromide. 3. Excise the gel slice containing the DNA of interest and place in a preweighed microcentrifuge tube. 4. Estimate the volume of the gel slice based upon the weight and determine the concentration of the DNA in the gel slice (e.g., 100 mg = 100 μl). Assume 100% recovery of the DNA. 5. Store excised band at 4 C until ready for use. 6. Add sterile distilled water to bring volume of the gel slice to 200 μl (200 mg). 7. Remelt the gel slice by heating to 68 C for 10 minutes. 8. Mix the melted gel slice by pipetting. 9. Remove a volume containing the quantity of DNA needed for restriction digestion. 10. Maintain the sample at 37 C to prevent gelling. 11. Dilute the restriction endonuclease with the appropriate reaction buffer. 12. Digest DNA at the appropriate temperature and time for your particular enzyme. 13. Stop the reaction by adding 30 μl of urea sample buffer to 50 μl of the digestion mixture and mixing. 14. Heat the sample to 65 C for 10 minutes if it has regelled. 15. Load the sample onto appropriate agarose gel for analysis. Materials Horizontal electrophoresis chamber Scalpel or razor blade Microcentrifuge tubes ( 1 ml) Water bath or heating block Reagents SeaPlaque GTG or NuSieve GTG Agarose 1X TAE (0.1 mm EDTA) Gel and Running Buffer Sterile distilled water GelStar or SYBR Green I Nucleic Acid Gel Stain or ethidium bromide Restriction endonuclease(s) Urea sample buffer 8% Ficoll (Type 400) Polymer 27 mm EDTA 0.27% bromophenol blue 5 M urea Adjust to ph 8.0 Caution: Materials and methods shown here present hazards to the user and the environment. Refer to the safety information on page 210 before beginning these procedures. References Hermann, R.G. and Whitfeld, P.R., Methods in Chloroplast Molecular Biology, Elsevier Biomedical Press, Hermann, R.G., et al., Gene 8: , Parker, R.C. and Seed, B., Meth. Enzymol. 65: , Peacock, A.C., et al., Anal. Biochem. 149: ,

121 Section V: In-Gel Reactions DNA Amplification in the Presence of Agarose Procedure 1 1. Electrophorese PCR products in low melting temperature agarose prepared in 1X TAE (0.1 mm EDTA) Buffer. 2. Briefly stain the gel with GelStar or SYBR Green I Gel Stain or ethidium bromide. 3. Excise a gel slice containing the template DNA and place into a preweighed microcentrifuge tube. 4. Store the gel slice at 4 C protected from light until ready for use. 5. Melt the agarose gel slice containing template DNA at 65 C for 10 minutes. 6. Dilute with 65 C, sterile distilled water to a final DNA concentration of 0.1 ng/μl. 7. Add a portion of the DNA into the amplification reaction mixture (do not exceed 1 ng DNA/reaction). 8. Combine the components of the reaction mixture. 9. Vortex the reaction mixture to mix the contents. 10. Spin briefly in a microcentrifuge. 11. Overlay the mixture with mineral oil if necessary. 12. Perform amplification reactions with conditions appropriate for the template DNA and primers. 13. Remove mineral oil. 14. Remelt the reaction mixture at 65 C prior to analyzing. Procedure 2 1. Follow steps 1-2 in Procedure Turn the gel upside down while it is on the UV transilluminator. 3. Stab the band of interest with a glass Pasteur pipette. When the pipette is removed from the gel, a small plug of agarose will be contained in the tip. 4. Remove the plug of agarose. 5. Add a portion of the plug of agarose to the amplification reaction. The plug does not require melting; it will melt during the first denaturing step. 6. Follow steps 8-14 in Procedure 1. Amplification Reaction Mixture If the reaction buffer does not contain magnesium ion, add sufficient amount for your template/primer. Component Volume (μl) FINAL Concentration 10X Reaction Buffer 5 1X dntp mix (1.25 mm each) μm each Primer #1, 10 μm μm or 1.0 μm Primer #2, 10 μm μm or 1.0 μm Template DNA + Agarose 5-10 (0.3 ng ng DNA) Polymerase 0.5 (2.5 units) Sterile distilled water to 50 μl Materials Horizontal electrophoresis chamber Scalpel or razor blade Vortex mixer Microcentrifuge Thin-walled microcentrifuge tube Microcentrifuge tubes ( 1 ml) Thermal cycler Reagents SeaPlaque GTG or NuSieve GTG Agarose 1X TAE (0.1 mm EDTA) Gel and Running Buffer DNA amplification kit Forward and reverse primers Sterile distilled water GelStar or SYBR Green I Nucleic Acid Gel Stain or ethidium bromide Caution: Materials and methods shown here present hazards to the user and the environment. Refer to the safety information on page 210 before beginning these procedures. In-Gel Reactions References Bjourson, A.J. and Cooper, J.E., Nucl. Acids Res. 20: 4675, Fors, L., et al., Nucl. Acids Res. 18: , Nichols, W.C., et al., Genomics 8: , Wilkie, T.M. and Simon, M.I., Methods: A Companion to Methods in Enzymology 2(1): 32-41,

122 Section V: In-Gel Reactions Restriction Digestion in the Presence of Agarose continued In-Gel Reactions References for other in-gel reactions Cycle Sequencing Fraser, G.J., Resolutions 9(3): 1, Gorman, K.B. and Steinberg, R.A., BioTechniques 7: , Kretz, K.A. and O Brein, J.S., Meth. Enzymol. 218: 72-79, Random Prime Labeling Daum, H.A. III, et al., BioTechniques. 11: , Feinberg, A.P. and Vogelstein, B., Anal. Biochem. 137: , Rose, E.A., et al., Cell. 60: , Soreum, H., et al., App. Environ. Microbiol. 56: Blunt-end Ligation Hemsley, A., et al., Nucl. Acids Res. 17: , Majumdar, D., et al., BioTechniques 7: , Murray, J.A.H., Nucl. Acids Res. 14: 10118, Translation & Transcription of RNA Brandt, T.L. and Hackett, P.B., Anal. Biochem. 135: , Gallitelli, D. and Hull, R., J. Virol. Methods 11: , Person, D.A. and Finn, O.J., BioTechniques 4: , Attaching Linkers Crouse, G.F., et al., Meth. Enzymol. 101: 78-89, Gap Filling Majumdar, D., et al., BioTechniques 7: ,

123 Section VI: Recovery of DNA from Agarose Gels Recovery of DNA from Agarose Gels In This Section Tips for Increasing DNA Recovery Efficiency from Agarose Gels 116 -Agarose Recovery of DNA from Agarose Gels 117 Electroelution of DNA from Agarose Gels 120 Phenol/Chloroform Extraction of DNA from Agarose Gels 122 "Modified Freeze/Squeeze" Extraction of DNA from Agarose Gels 123 Ethanol Precipitation of DNA Recovered from Agarose Gels 124 References

124 Section VI: Recovery of DNA from Agarose Gels Tips for Increasing DNA Recovery Efficiency from Agarose Gels Recovery of DNA from Agarose Gels Introduction This section discusses various tips which will increase the efficiency of recovery of DNA from agarose gels. These tips and recommendations can be applied to all recovery techniques. The section is divided into the following topics: Choosing the appropriate agarose Choosing the appropriate electrophoresis buffer Gel casting and DNA loading tips Staining and recovery tips Choosing the appropriate agarose for recovery When recovering DNA, the choice of agarose is one of the most important factors. To avoid recovery altogether, one can choose to perform in-gel reactions. Lonza offers Genetic Technology Grade (GTG ) Products that are specially prepared for demanding molecular biology applications. Lonza s GTG Product quality tests go beyond standard nuclease assays to include enzymatic performance measurements. Our additional testing provides a more realistic index of overall product quality and reliability. You no longer need to screen agarose lots to find those which yield biologically active DNA. Lonza agaroses and compatible recovery techniques SeaKem GTG Agarose SeaPlaque GTG Agarose NuSieve GTG Agarose MetaPhor Agarose SeaPlaque Agarose Cat. No. In-Gel -Agarase Phenol/ Chloroform Recovery Columns Electroelution Buffer types When recovering DNA from agarose gels, 1X Tris-acetate (TAE) Buffer is recommended for electrophoresis. Casting and DNA loading tips Prepare the gel in 1X TAE Buffer Do not cast the gel with ethidium bromide Cast a gel 3 mm - 4 mm thick Use a combs 1 mm thick Load no more than 100 ng of DNA per band Staining and recovery tips When recovering DNA from agarose gels, we recommend the following: Stain the gel for minutes Destain the gel in distilled water for two, 20-minute washes Do not expose the DNA to UV light for any longer than 1 minute; long exposure of DNA to UV light can nick the DNA The addition of 1 mm guanosine or cytidine to the gel and electrophoresis buffer is effective in protecting DNA against UV-induced damage Cut the smallest gel slice possible It is possible to avoid staining samples which will be used for recovery by running an additional lane containing a small amount of your sample immediately next to the molecular weight marker. However, DNA is damaged by UV light in the absence of ethidium bromide so keep exposure to UV light as brief as possible. Cut the lanes containing the marker and the small amount of the sample from the rest of the gel and stain. To recover the preparative loading, line up the stained portion of the gel with the unstained portion. Check by placing on UV transilluminator and cut out the area that lines up with your sample on the unstained portion of the gel. Freeze/ Squeeze The FlashGel System for Recovery, (page 20) offers a non-uv alternative for DNA recovery. Reference Grundemann, D. and Schomig, E., BioTechniques 21(5): ,

125 Section VI: Recovery of DNA from Agarose Gels ß-Agarase Recovery of DNA from Agarose Gels Introduction ß-Agarase is an enzyme that digests the polysaccharide backbone of agarose to alcohol-soluble oligosaccharides. DNA in a low melting temperature agarose gel can be recovered after the gel is melted and digested with this enzyme. The remaining oligosaccharides will not gel or interfere with subsequent DNA manipulations such as cloning, labeling, restriction digestion or sequencing. Lonza s ß-Agarase is free of any detectable DNase, RNase and phosphatase activities. ß-Agarase recovery is particularly useful for recovering large DNA (>10 kb) which could be sheared by other methods of recovery. Compatible agaroses SeaPlaque GTG Agarose (certified and tested for the recovery of DNA) NuSieve GTG Agarose (certified and tested for the recovery of DNA) SeaPlaque Agarose Tips Transfer no more than 200 mg of the agarose gel to a microcentrifuge tube for equilibration with digestion buffer Completely melt the gel slice prior to the addition of enzyme Ethanol precipitations should be incubated at room temperature with ammonium acetate rather than sodium acetate to decrease the likelihood of coprecipitation of agarose-oligosaccharides with the DNA Polynucleotide kinase is inhibited by 7 mm ammonium ion; use sodium acetate for your precipitation if you will be phosphorylating your DNA after recovery The enzyme preparation retains full activity for several hours at 45 C; however, it will gradually lose activity upon longer incubations; for this reason, incubate at 40 C for overnight digestions For efficient recovery of small nucleic acids (<500 bp) and/or very dilute samples (<0.05 μg/ml), we recommend either carrier trna or nuclease-free glycogen be added; in addition, overnight precipitation at room temperature can be helpful The enzyme preparation has maximum activity between ph 6-7 and is relatively unaffected by salt concentrations between 0.1 M and 0.25 M; the equilibration of the gel slice with ß-Agarase Buffer is necessary to provide the enzyme with optimal buffer conditions; this equilibration is more important for gels prepared in TBE than in TAE Buffer due to the greater buffering capacity of TBE Buffer Ethanol precipitate the DNA at room temperature or on ice. The most effective DNA precipitation can be achieved at 0 C to 22 C, rather than at -20 C to -70 C. At higher temperatures, yields are more consistent and precipitation of oligosaccharides will be avoided ß-Agarase unit definition One unit of Lonza ß-Agarase will completely digest 200 mg of a molten 1% SeaPlaque GTG Agarose gel which has been dialyzed in 1X ß-Agarase Buffer in 1 hour at 40 C; similar activities are obtained for other low melting temperature agaroses such as NuSieve GTG Agarose. Concentration: 1 unit per μl Recovery of DNA from Agarose Gels 117

126 Section VI: Recovery of DNA from Agarose Gels ß-Agarase Recovery of DNA from Agarose Gels continued Recovery of DNA from Agarose Gels Procedure for ß-Agarase digestion BEFORE YOU BEGIN: Set one water bath or heating block for 70 C. Set another water bath or heating block for 45 C. 1. Electrophorese DNA in a low melting temperature agarose gel (such as SeaPlaque, SeaPlaque GTG, MetaPhor or NuSieve GTG Agarose) which has been prepared in TAE or TBE Buffer. 2. Briefly stain the gel with GelStar or SYBR Green I Nucleic Acid Stain or ethidium bromide. 3. Photograph the gel. 4. Excise a gel slice containing the DNA of interest. 5. Place the gel slice containing the DNA sample into a microcentrifuge tube. 6. Equilibrate the gel slice with 10 volumes of 1X ß-Agarase Buffer for 60 minutes at room temperature OR follow steps 6A - 6D. 6a. Melt the gel slice at 70 C for 15 minutes. Make sure that the gel slice is completely melted as the enzyme preparation will not digest agarose in the gelled state. 6b. Add 2 μl of the 50X ß-Agarase buffer to approximately 100 μl of melted gel solution. 6c. Mix the solution. 6d. Proceed to steps 9, 10 and Discard the buffer. 8. Melt the gel slice at 70 C (approximately 15 minutes). Make sure the gel slice is completely melted as the enzyme preparation will not digest agarose in the gelled state. 9. Cool the melted agarose solution to 45 C. 10. Add 1 unit of ß-Agarase for 200 mg (approximately 200 μl) of 1% agarose gel (add proportionally more or less of the enzyme preparation for larger or smaller gel slices, or with higher or lower agarose concentrations, respectively). Skip to step If you have followed the protocol in steps 6A - 6D, the amount of enzyme will need to be adjusted. If the gel was Then you will prepared with... need to add... TAE Buffer TBE Buffer Twice the amount of enzyme Seven times the amount of enzyme 12. Mix. 13. Incubate at 45 C for 60 minutes. 14. If the DNA is 30 kb... Place the solution at -20 C for 15 minutes. Centrifuge the solution for 15 minutes at 4 C. Continue with Step 15 OR The sample can be concentrated without ethanol precipitation by centrifugation in a molecular weightcut-off-spin column (e.g., Amicon s Microcon Microconcentrator). If the DNA is >30 kb... The DNA/ß-Agarase solution may be used directly without precipitation for subsequent enzymatic manipulations. OR The sample can be concentrated without ethanol precipitation by centrifugation in a molecular weightcut-off-spin column (e.g., Amicon s Microcon or Centricon Microconcentrators). 15. Transfer the supernatant to a new microcentrifuge tube leaving behind any undigested agarose in the pellet. 16. Add ammonium acetate to the supernatant to a final concentration of 2.5 M. 17. Add 2 to 3 volumes of 100% ethanol. 18. If the DNA is 0.05 μg/ml... Precipitate at room temperature for 24 hours OR Add 10 μg of RNA. If the DNA is >0.05 μg/ml... Precipitate at room temperature for 30 minutes. 19. Collect the precipitate by centrifugation. Caution: Materials and methods shown here present hazards to the user and the environment. Refer to the safety information on page 210 before beginning these procedures. 118

127 Section VI: Recovery of DNA from Agarose Gels ß-Agarase Recovery of DNA from Agarose Gels continued Materials Microcentrifuge tubes ( 1 ml) Two water baths or heating blocks Scalpel or razor blade Microcentrifuge Reagents SeaPlaque, SeaPlaque GTG, NuSieve GTG or MetaPhor Agarose GelStar or SYBR Green I Nucleic Acid Gel Stain or ethidium bromide ß-Agarase buffer Supplied as a 50X concentrate; 1X concentration is: 40 mm Bis Tris/HCl 40 mm NaCl, 1 mm EDTA (ph 6.0) ß-Agarase Enzyme 10 M ammonium acetate 100% ethanol Recovery of DNA from Agarose Gels Caution: Materials and methods shown here present hazards to the user and the environment. Refer to the safety information on page 210 before beginning these procedures. References Chong, S. and Garcia, G.A., BioTechniques 17: , Crouse, J. and Amorese, D., GIBCO-BRL Focus 9: 3-5, Lamb, B.T., et al., Nature Genetics 5: 22-29, Morrice, L.M., et al., Can. J. Microbiol. 3: , Richardson, C.C., The Enzymes, 3rd Edition, Academic Press, Zeugin, J.A. and Hartley, J.L., GIBCO-BRL Focus 7(4): 1-2,

128 Section VI: Recovery of DNA from Agarose Gels Electroelution of DNA from Agarose Gels Recovery of DNA from Agarose Gels Introduction Electroelution is a reliable and consistent recovery method. The recovered DNA is suitable for a wide range of applications. For small fragments, a typical yield would be 50-85%, but as fragment length increases, yield can drop as low as 20%. The procedure below describes the electroelution of DNA into dialysis bags and has been adapted from Sambrook, et al. Alternatively, if you have a commercially available apparatus, follow the manufacturer s instructions. Compatible agaroses SeaKem GTG Agarose (certified and tested for the recovery of DNA) SeaPlaque GTG Agarose (certified and tested for the recovery of DNA) NuSieve GTG Agarose (certified and tested for the recovery of DNA) SeaPlaque Agarose NuSieve 3:1 Agarose MetaPhor Agarose Tips Electrophorese DNA in 1X TAE Buffer Have 1 μg of DNA in your band of interest Minimize exposure of DNA to UV light for no more than 1 minute Cut the smallest gel slice possible Materials Scalpel or razor blade Spatula Dialysis tubing Dialysis tubing clips Electrophoresis chamber Hand-held, long-wavelength ultraviolet lamp Disposable plastic tube Pasteur pipette Reagents 1X TAE Buffer 2% w/v sodium bicarbonate, 1 mm EDTA ph 8.0, prepare in distilled water 1mM EDTA, ph 8.0 Distilled water GelStar or SYBR Green I Nucleic Acid Gel Stain or ethidium bromide Caution: Materials and methods shown here present hazards to the user and the environment. Refer to the safety information on page 210 before beginning these procedures. Procedure for preparing dialysis tubing 1. Cut the tubing into 10 cm to 20 cm long pieces. 2. Boil for 10 minutes in a large volume of 2% sodium bicarbonate/1 mm EDTA, ph Rinse the tubing with distilled water. 4. Boil for 10 minutes in 1 mm EDTA, ph Cool the tubing. 6. Store at 4 C submerged in 1 mm EDTA, ph

129 Section VI: Recovery of DNA from Agarose Gels Electroelution of DNA from Agarose Gels continued Procedure for electroeluting DNA from agarose gels 1. Electrophorese DNA in an agarose gel. 2. Stain the gel with GelStar or SYBR Green I Nucleic Acid Stain or ethidium bromide. 3. Locate the band of interest using a UV light source. Minimize exposure of DNA to UV light to less than 1 minute. 4. Excise the band of interest using a scalpel or razor blade. 5. Wet a spatula with 1X TAE Buffer. 6. Place the agarose slice containing the DNA on a wetted spatula or a scalpel. 7. Photograph the gel for a record of which band was eluted. 8. Seal one end of treated dialysis tubing with a dialysis clip. 9. Fill the bag to the top with 1X TAE Buffer. 10. Transfer the slice of agarose into the bag with the spatula. 11. Allow the slice of agarose to sink to the bottom of the bag. 12. Remove the buffer from the dialysis bag, leaving just enough to keep the gel slice in constant contact with the buffer. 13. Clip the dialysis bag above the gel slice, avoiding air bubbles. 14. Place a shallow layer of 1X TAE Buffer in an electrophoresis chamber. 15. Immerse the bag in the electrophoresis chamber. 16. Pass electric current through the bag (4-5 V/cm for 2-3 hours). During this time, the DNA is eluted out of the gel and onto the inner wall of the dialysis tubing. This process can be monitored with a hand-held, long-wavelength UV lamp. Expose to UV light briefly. 17. Reverse the polarity of the current for 1 minute. This will remove the DNA from the wall of the bag. 18. Recover the bag from the electrophoresis chamber. 19. Gently massage the side of the bag with gloved fingers where the DNA has accumulated. This will remove the DNA from the wall of the bag. This process can be monitored with a hand-held UV lamp. Expose to UV light briefly. 20. Open the dialysis bag. 21. Transfer all of the buffer surrounding the gel slice to a plastic tube. 22. Wash out the bag with a small amount of 1X TAE Buffer. 23. Transfer the solution to the plastic tube. 24. Remove the gel slice from the bag. 25. OPTIONAL: Stain the slice with GelStar or SYBR Green I Nucleic Acid Stain or ethidium bromide to ensure all the DNA has been eluted from the slice. 26. Purify the DNA from 1X TAE Solution using phenol/ chloroform extractions.* * After electroelution, it is recommended that the DNA is further purified with a phenol/chloroform extraction followed by ethanol precipitation. Oligosaccharides and other contaminants (found in low-grade agarose) can copurify with the DNA. Phenol extractions will remove any oligosaccharides, avoiding their coprecipitation during ethanol precipitations. Recovery of DNA from Agarose Gels References Bostian, K.A., et al., Anal. Biochem. 95: , Dretzen, G., et al., Anal. Biochem. 112: , Girvitz, S.D., et al., Anal. Biochem. 106: , Henrich, B., et al., J. Biochem. Biophys. Methods 6: , Ho, N.W.Y., Electrophoresis 4: , Smith, H.O., Meth. Enzymol. 65: , Strongin, A.Y., et al., Anal. Biochem. 79: 1-10, Tabak, H.F. and Flavell, R.A., Nucl. Acids Res. 5: ,

130 Section VI: Recovery of DNA from Agarose Gels Phenol/Chloroform Extraction of DNA from Agarose Gels Recovery of DNA from Agarose Gels Compatible agaroses Tips SeaPlaque GTG Agarose (certified and tested for the recovery of DNA) NuSieve GTG Agarose (certified and tested for the recovery of DNA) SeaPlaque Agarose Recovery failures when extracting DNA from agarose using phenol/chloroform most often result from either extracting too large a piece of agarose, or precipitating agarose along with the DNA at the ethanol precipitation step. To address these difficulties, we recommend the following: No more than 200 mg (200 μl) of agarose should be extracted in a single tube; if your gel slice containing the DNA is larger than this, separate it into smaller pieces, then combine the extracted solutions prior to ethanol precipitation Ethanol precipitation of agarose can be avoided by chilling the extracted solution on ice for 15 minutes, then centrifuging the sample(s) in a cold room for 15 minutes at maximum speed in a microcentrifuge prior to adding salts and ethanol. The supernatant is then carefully decanted, and the DNA in the supernatant is precipitated following standard protocols Not useful for large DNA (>10kb). Vortexing will shear the DNA Procedure This protocol has been adapted from Sambrook, et al. 1. Electrophorese DNA in a low melting temperature agarose gel prepared in 1X TAE Buffer. 2. Set a water bath for 67 C. 3. Prewarm the TE at 67 C. 4. Excise the gel fragment containing the DNA. 5. Place the DNA slice in a microcentrifuge tube. 6. Estimate the volume of the slice. If the slice is significantly greater than 200 mg, break the agarose slice into smaller pieces and place each agarose piece in a separate microcentrifuge tube. 7. Melt the gel slice at 67 C for 10 minutes. 8. Add the appropriate volume of TE Buffer (prewarmed to 67 C) so that the final concentration of agarose is 0.5%. 9. Maintain the samples at 67 C until you are ready to phenol extract. 10. Add an equal volume of buffer-equilibrated phenol. 11. All subsequent steps can be done at room temperature. 12. Vortex for 15 seconds. 13. Centrifuge at maximum speed in a microcentrifuge for 3 minutes. 14. Carefully remove the top aqueous phase. The interface of white debris is the agarose, which can contain some trapped DNA. This can be back-extracted with TE to maximize yield. 15. Place aqueous phase in a clean microcentrifuge tube. 16. Repeat steps Add an equal volume of phenol/chloroform to the aqueous phase. 18. Vortex for 15 seconds. 19. Centrifuge at maximum speed in a microcentrifuge for 3 minutes. 20. Remove the aqueous phase and place in a clean microcentrifuge tube. 21. Repeat the extraction with an equal volume of chloroform. 22. Remove the aqueous phase and place in a clean microcentrifuge tube. 23. Chill the aqueous phase for 15 minutes on ice. 24. Centrifuge in a microcentrifuge at high speed for 15 minutes at 4 C. 25. Carefully decant the supernatant into a clean microcentrifuge tube. 26. Ethanol precipitate the DNA in the supernatant following standard protocols. 122

131 Section VI: Recovery of DNA from Agarose Gels Phenol/Chloroform Extraction of DNA from Agarose Gels Continued Materials Water bath set to 67 C Microcentrifuge tubes ( 1 ml) Ice bucket and ice Vortex mixer Microcentrifuge Scalpel or razor blade References Benson, S.A., BioTechniques 2: 66-67, Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Section VI: Recovery of DNA from Agarose Gels "Modified Freeze/Squeeze" Extraction of DNA from Agarose Gels Reagents SeaPlaque GTG, NuSieve GTG or SeaPlaque Agarose TE Buffer warmed to 67 C Buffer-equilibrated penol Chloroform Phenol/chloroform (1:1) Caution: Materials and methods shown here present hazards to the user and the environment. Refer to the safety information on page 210 before beginning these procedures. Recovery of DNA from Agarose Gels Introduction This protocol is relatively fast and will work with low and standard melting temperature agaroses. However, like many recovery techniques, the DNA yield decreases with increasing DNA size, but should be approximately 50% for DNA 5 kb. Compatible agaroses SeaKem GTG Agarose (certified and tested for the recovery of DNA) SeaPlaque GTG Agarose (certified and tested for the recovery of DNA) NuSieve GTG Agarose (certified and tested for the recovery of DNA) SeaPlaque Agarose MetaPhor Agarose NuSieve 3:1 Agarose SeaKem Gold Agarose Procedure This protocol has been adapted from Benson. For more detailed protocols on phenol chloroform extractions, refer to the previous section. 1. Electrophorese DNA in an agarose gel prepared in 1X TAE Buffer. References Benson, S.A., BioTechniques 2: 66-67, Polman, J.K. and Larkin, J.M., Biotechnology Techniques 3: , Tautz, D. and Renz, M., Anal. Biochem. 132: 14-19, Excise gel slice containing DNA. 3. Place in a microcentrifuge tube. 4. Estimate volume based on weight. 5. Mash the gel slice with the stir rod. 6. Add an equal volume of buffer-equilibrated phenol. 7. Vortex for 10 seconds. 8. Freeze at -70 C for 5 to 15 minutes. 9. Centrifuge for 15 minutes at room temperature. 10. Remove the supernatant which contains the DNA. 11. Place the supernatant, in a clean microcentrifuge tube. 12. Phenol/chloroform extract the supernatant. 13. Follow with a chloroform extraction. 14. Ethanol precipitate following standard procedures. Materials Microcentrifuge tubes (" 1 ml) Ice bucket and ice Vortex mixer Microcentrifuge Scalpel or razor blade Glass or plastic stir rod Reagents TE Buffer warmed to 67 C Buffer equilibrated phenol Chloroform Phenol/chloroform (1:1) 123

132 Section VI: Recovery of DNA from Agarose Gels Ethanol Precipitation of DNA Recovered from Agarose Gels Recovery of DNA from Agarose Gels Introduction This method is compatible with all the recovery techniques listed in this chapter. Tips Prior to adding salts and ethanol, precipitation of agarose can be avoided by chilling the supernatant on ice for 15 minutes, then centrifuging the sample(s) in a cold room for 15 minutes at maximum speed in a microcentrifuge. The supernatant is then carefully decanted, and the DNA in the supernatant is ethanol precipitated following standard protocols Ethanol precipitations should be incubated at room temperature with ammonium acetate rather than sodium acetate in order to decrease the likelihood of coprecipitation of agarose-oligosaccharides with the DNA or RNA Materials Ice bucket and ice Vortex mixer Microcentrifuge Vacuum gel dryer Microcentrifuge tubes ( 1 ml) Reagents 100% ethanol (ice cold) 70% ethanol (room temperature) TE Buffer, ph M ammonium acetate Procedure 124 This protocol has been adapted from Sambrook, et al. 1. Chill the supernatant on ice for 15 minutes. 2. Centrifuge the sample(s) in a cold room for 15 minutes at maximum speed in a microcentrifuge. 3. Carefully decant the supernatant. 4. Place in a clean microcentrifuge tube. 5. Measure the volume of the sample. 6. Add 0.2 volumes of 10 M ammonium acetate to the sample. 7. Add 2 volumes of 100% ice-cold ethanol. 8. Briefly vortex. 9. Store the mixture for 30 minutes to overnight at room temperature. 10. Centrifuge for 30 minutes at 12,000 rpm. 11. Decant the supernatant. 12. Wash the pellet three times with 70% ethanol. 13. Allow to air-dry at room temperature on the bench top. 14. Dry under vacuum for 5-10 minutes. 15. Dissolve the DNA in TE Buffer. References Life Technologies, Inc., GIBCO-BRL Focus 7(4): 1-2, Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, References for other recovery methods References for Electrophoresis onto DEAE-cellulose membrane Dretzen, et al., Anal. Biochem. 112: 295, Girvitz, et al., Anal. Biochem. 106: 492, Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, Winberg, G. and Hammarskjold, M.L., Nucl. Acids Res. 8: , Yang, R.C.A., et al., Meth. Enzymol. 68: , Zassenhaus, H.P., et al., Anal. Biochem. 25: , References for Passage through DEAE-sephacel Sambrook, J., et al., Molecular Cloning : A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, References for Recovery using glass beads Ausubel, F.M., et al., Current Protocols in Molecular Biology, Red Book, Wiley & Sons, Chen, C.W. and Thomas, C.A., Anal. Biochem. 101: , Vogelstein, B. and Gillespie, D., Proc. Natl. Acad. Sci. 76: , General References Ausubel, F.M., et al., Current Protocols in Molecular Biology, Red Book, Wiley & Sons, Hengen, P.N., Methods and Reagents; Recovering DNA from Agarose Gels. TIBS 19, Maniatis, T., et al., Molecular Cloning: A Laboratory Manual, Cold Spring Harbor, Cold Spring Harbor Laboratories, Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, 1989.

133 Section VII: Separation of DNA in Polyacrylamide Gels Separation In DNA in Polyacrylamide Gels In This Section Separating DNA in Polyacrylamide Gels 126 Detecting DNA in Polyacrylamide Gels 127 Recovery of DNA in Polyacrylamide Gels 129 References

134 Section VII: Separation of DNA in Polyacrylamide Gels Separating DNA in Polyacrylamide Gels Separation of DNA in Polyacrylamide Gels Overview Polyacrylamide gels can separate DNA that differs by 0.2% in length, well beyond the resolving capabilities of agarose (2% difference in DNA length). Another advantage to using polyacrylamide gels is that they can accommodate large amounts of DNA (up to 10 μg) without any loss in resolution. Depending upon the application, TBE gels can be prepared as denaturing or nondenaturing gels. Lonza offers PAGEr Precast TBE Gels for DNA separation. Refer to page 17 for information. Applications Denaturing gels: concentrations range from 8-20% Oligonucleotide purification Separation of single-stranded DNA Isolate radiolabeled DNA probes S1 nuclease assay DNA footprinting RNase protection assays Nondenaturing gels: concentrations range from 3-20% Separation of di-nucleotide repeats Separation of DNA ranging from 20 bp bp in length Study DNA-Protein interactions (Gel Shift Assays) TBE is commercially available as 5X or 10X solutions (Lonza AccuGENE TBE Buffer). Alternatively, it can be prepared as follows: 10X TBE Stock Solution (890 mm Tris base, 890 mm Boric acid, 20 mm EDTA) 1X = 89 mm Tris base, 89 mm Boric acid, 2 mm EDTA) g Tris base 55.0 g Boric acid 7.44 g Na Adjust volume to 1 liter with distilled water Filter through a 0.45 μm filter ph adjustment is not necessary Suggested Polyacrylamide Concentrations Nondenaturing polyacrylamide gels Gel Percentage Fragment Sizes 3.5% 1,000 bp - 2,000 bp 5.0% 80 bp bp 6.0% 75 bp - 2,000 bp 8.0% 60 bp bp 10.0% 30 bp - 1,000 bp 12.0% 40 bp bp 15.0% 25 bp bp 20.0% 5 bp bp 4-20% 10 bp - 2,000 bp Buffers for Electrophoresis To ensure adequate buffering power during vertical electrophoresis, TBE Buffer is used for polyacrylamide gel electrophoresis at a working strength of 1X. Lower dilutions of the buffer or the use of TAE Buffer may cause gels to overheat and result in band smiling throughout the gel. 126

135 Section VII: Separation of DNA in Polyacrylamide Gels Detecting DNA in Polyacrylamide Gels Detecting DNA in Polyacrylamide Gels with GelStar or SYBR Green Nucleic Acid Gel Stains GelStar and SYBR Green Nucleic Acid Gel Stains are highly sensitive ffiuorescent stains for detecting nucleic acids. These stains provide high sensitivity detection of double-stranded or single-stranded DNA. See table below for a comparison of staining sensitivities and limits of detection. Stain ssdna dsdna Native RNA GelStar Stain 25 pg 20 pg 10 ng SYBR Green I Stain pg SYBR Green II Stain 60 pg 15 ng Ethidium bromide 350 pg 100 pg 30 ng Tips for Staining Gels with GelStar and SYBR Green Nucleic Acid Gel Stains The powder used on some laboratory gloves may contribute to background ffiuorescence. We recommend using powder-free gloves and rinsing gloves prior to handling gels Fibers shed from clothing or lab coats may be ffiuorescent; be cautious when handling gels Staining of nucleic acids with these dyes has minimal impact on blotting efficiency. To ensure efficient hybridization, use of prehybridization and hybridization solutions containing % SDS is important to remove stain retained during transfer These stains can be removed from nucleic acids by ethanol precipitation. Isopropyl alcohol precipitation is less effective at removing the dye; butyl alcohol extraction, chloroform extraction and phenol do not remove the dye efficiently Allow time for the stock solution to thaw completely. Removal of stain from partially thawed solutions will result in depletion of stain over time. These stains may be diluted in most common electrophoresis buffers with a ph range from or in TE Buffer. Staining solutions prepared in water or in buffer with a ph below 7.0 or above 8.5 are less stable and show reduced staining efficiency Prepare and store the stain in polypropylene containers such as Rubbermaid containers or pipette-tip box lids. The stain may adsorb to glass surfaces and some plastic surfaces, particularly if the surfaces carry residues of anionic detergents or reagents As an alternative to the protocol presented for staining gels on the cassette plate, smaller gels such as mini gels may be removed from both plates then stained using the protocol for post-staining agarose gels found in Section IV. Treatment of one plate with a release agent, such as Gel Slick Solution, increases the ease of separating the glass plates while keeping the gel in place on the other plate for staining Handling or compression of gels (particularly polyacrylamide-type gels) can lead to regions of high background after staining. If possible, gels should not be handled directly; use a spatula (or other tool) and a squirt bottle to slide the gel off the plates and into the stain or onto the light box Procedure for Staining Incorporating these dyes into the gel or prestaining the nucleic acid in a vertical format is not recommended. The dye binds to glass or plastic plates and DNA may show little to no signal. Gels should be post-stained as described below. 1. Remove the concentrated stock solution from the freezer and allow the solution to thaw at room temperature 2. Spin the solution in a microcentrifuge to collect the dye at the bottom of the tube 3. Dilute the 10,000X concentrate to a 1X working solution for DNA and a 2X working solution for RNA, in a ph buffer, in a clear plastic polypropylene container 4. Open the cassette, and leave the gel in place on one plate 5. Place the plate, gel side up, in a staining container 6. Gently pour the stain over the surface of the gel; a disposable pipette may be used to help distribute the stain evenly over the gel surface 7. Stain the gel for 5-15 minutes. No destaining is required 8. Remove the gel from the staining solution and view with a 300 nm UV transilluminator, Dark Reader Transilluminator or CCD imaging system. 127 Separation of DNA in Polyacrylamide Gels

136 Section VII: Separation of DNA in Polyacrylamide Gels Detecting DNA in Polyacrylamide Gels continued Separation of DNA in Polyacrylamide Gels For highest sensitivity, the gel should be carefully removed from the plate and placed directly on the transilluminator or scanning stage. Alternatively, if a relatively low uorescence plate is used, the results may be visualized by placing the gel and plate gel side down on the transilluminator and photographing or by scanning the gel directly on the plate. NOTE: More detailed information on photographing gels and decontaminating staining solutions is described in Detecting DNA with GelStar or SYBR Green I Stains (see Section IV). Detecting DNA in Polyacrylamide Gels with Ethidium Bromide The procedure for post-staining DNA in polyacrylamide gels with ethidium bromide is identical to the procedures used for post-staining agarose gels. Follow the procedures described in Detecting DNA with Ethidium bromide (see Section IV). Materials Clear polypropylene container (e.g., Rubbermaid Recycling #5 Plastics) Microcentrifuge UV transilluminator Photographic Filter for GelStar Stain or SYBR Green Stain (Wratten #15 or Wratten #9 Filter respectively) Reagents GelStar Nucleic Acid Gel Stain or SYBR Green I or II Nucleic Acid Gel Stain Buffer between ph (TBE or TE) Caution: Materials and methods shown here present hazards to the user and the environment. Refer to the safety information on page 210 before beginning these procedures. 128

137 Section VII: Separation of DNA in Polyacrylamide Gels Recovery of DNA in Polyacrylamide Gels The two primary methods for recovering DNA from polyacrylamide gels are the Crush and Soak method or electroelution. Described below is the Crush and Soak procedure. The procedure for electroeluting DNA from polyacrylamide gels is similar to the procedures used for agarose gels with one exception; 0.5X TBE Buffer should be used rather than TAE Buffer. Follow the procedures described in Electroelution of DNA from Agarose Gels (see Section VI). Tips for increasing DNA recovery from polyacrylamide gels When recovering DNA from polyacrylamide gels, we recommend the following: Stain the gel for no more than 15 minutes If ethidium bromide staining, destain the gel in distilled water for two, 20-minute washes Do not expose the DNA to UV light for any longer than 1 minute. Longer exposures may result in DNA nicking Cut the smallest gel slice possible If recovering small amounts of DNA, the addition of 10 μg of carrier RNA prior to ethanol precipitation may improve recovery yields Crush and soak procedure 1. While the gel is on the transilluminator, cut out the band of interest using a razor blade or scalpel. Cut the smallest size gel slice possible. NOTE: If the gel has been covered with plastic wrap, do not remove the plastic wrap before cutting. 2. Peel the small piece of gel containing the DNA from the plastic wrap. 3. Transfer the gel slice to a microfuge tube. 4. Crush the gel slice against the wall of the microfuge tube with the disposable pipette tip. 5. Add 1-2 volumes of elution buffer to the gel slice (e.g., if the estimated gel slice volume is 200 μl add 200 μl μl of elution buffer). 6. Incubate the tube at 37 C on a rotating wheel or rotary platform. For fragments less than 500 bp incubate for 3-4 hours. For fragments greater than 500 bp incubate for hours. 7. Centrifuge the samples at 12,000 rpm for 1 minute at 4 C. 8. Transfer the supernatant to a fresh microfuge tube, being careful to avoid transferring fragments of polyacrylamide. 9. Add 0.5 volumes of elution buffer to the pellet of polyacrylamide. 10. Vortex briefly. 11. Centrifuge at 12,000 rpm for 1 minute at 4 C. 12. Combine the two supernatants. 13. Remove any remaining polyacrylamide by passing the supernatant through a disposable plastic column or a syringe barrel containing a Whatman GF/C Filter or packed siliconized glass wool. 14. Add 2 volumes of cold ethanol. 15. Store the solution on ice for 30 minutes. 16. Recover the DNA by centrifugation at 12,000 rpm for 10 minutes at 4 C. 17. Decant the supernatant. 18. Redissolve the DNA in 200 μl of AccuGENE 1X TE Buffer, ph Add 25 μl of AccuGENE 3 M Sodium Acetate, ph Repeat steps Rinse pellet once with 70% ethanol. 22. Add 10 μl of AccuGENE 1X TE, ph 7.6 to pellet and dissolve. Separation of DNA in Polyacrylamide Gels 129

138 Section VII: Separation of DNA in Polyacrylamide Gels Recovery of DNA in Polyacrylamide Gels continued Separation of DNA in Polyacrylamide Gels Materials Scalpel or razor blade Microfuge tube Disposable pipette tip Rotary wheel or platform 37 C oven Microcentrifuge at 4 C Vortex Disposable plastic column or a syringe barrel containing a Whatman GF/C Filter or packed siliconized glass wool Ice Reagents Elution buffer (3.85 g ammonium acetate, g magnesium acetate, 0.2 ml AccuGENE 0.5 M EDTA Solution, 1.0 ml AccuGENE 10% SDS Solution, fill to 100 ml with distilled water) 100% and 70% ethanol AccuGENE 1X TE, ph 7.6 AccuGENE 3M Sodium Acetate, ph References Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Rickwood, D. and Hames, B.D., Gel Electrophoresis of Nucleic Acids: A Practical Approach, 2nd edition, IRL Press, Ausubel, F.M., et al., Short Protocols in Molecular Biology, 4th edition, John Wiley & Sons, Inc., Brown, T.A., Essential Molecular Biology: A Practical Approach, volume 1, 2nd edition, Oxford University Press, 2000.

139 Section VIII: Separation of RNA in Agarose Gels Separation of RNA in Agarose Gels In This Section Introduction 132 Preparation of RNA Samples 132 Buffers for RNA Electrophoresis 134 Electrophoresis of RNA 134 Detection of RNA in Agarose Gels with GelStar or SYBR Green II Gel Stains 138 Detecting RNA with Ethidium Bromide 142 Nothern Blotting 143 FlashGel System for RNA Analysis 147 References

140 Section VIII: Separation of RNA in Agarose Gels Introduction Separation of RNA in agarose gels is used for a number of different purposes, including Northern blots to monitor RNA expression levels, checking RNA integrity and sizeselection of RNA for cloning experiments. Separation of RNA based on fragment length requires conditions that are different from DNA analysis. These include sample preparation, the use of sample and gel denaturants, electrophoresis buffers, and visualization. Denaturing systems The purpose of the experiment and the size of the RNA being separated are the primary drivers in determining which denaturing system to use. The most frequently used denaturants for RNA agarose gel electrophoresis are formaldehyde, formaldehyde/formamide, and glyoxal plus DMSO. In each system, the denatured RNA migrates through the agarose gel in a linear relation to the log of its molecular weight (similar to DNA). The most efficient RNA denaturant is methylmercury hydroxide. Because of the hazards associated with this denaturant, it is the least used system for RNA analysis. The resolving powers of the glyoxal/dmso and the formaldehyde buffer systems are nearly identical. For detection by Northern analysis, glyoxal/dmso denaturant is preferable because these gels tend to produce sharper bands than the formaldehyde system. Glyoxal gels require more care to run than formaldehyde gels and because of the lower buffering capacity of glyoxal, these gels must be run at lower voltages than gels containing formaldehyde. Glyoxal gels require a phosphate electrophoresis buffer and the buffer must be recirculated during electrophoresis. If the ph of the buffer rises above 8.0, glyoxal dissociates from RNA, causing the RNA to renature and migrate in an unpredictable manner. For staining purposes, either denaturant can be used. Ethidium bromide, GelStar Nucleic Acid Gel Stain and SYBR Green II Gel Stain bind formaldehyde-denatured RNA more efficiently than glyoxal-denatured RNA. Glyoxal denaturant can interfere with binding of the stain, but gel backgrounds are often lower in these gels than with formaldehyde-denatured gels. It is important to minimize RNase activity when running agarose gels by following certain precautions. There are several agents on the market that effectively remove RNase s or consult Sambrook, et al. Section VIII: Separation of RNA in Agarose Gels Preparation of RNA Samples Separation of RNA in Agarose Gels 132 Which sample denaturation method to choose depends on the final goal of the experiment and the secondary structure of the RNA. There are several procedures to choose from, the most useful of which are described here. Any sample denaturation method can be used with any of the gel buffering systems. If simply checking the integrity of cellular RNA, no sample denaturation is necessary and TAE or TBE Buffer can be used. Common denaturants Formamide Formaldehyde Glyoxal Application Retain biological activity Sample recovery Northern blotting; significant secondary structure Caution: Materials and methods shown here present hazards to the user and the environment. Refer to the safety information on page 210 before beginning these procedures. Formamide-only denaturation Formamide denaturation is suitable for almost all RNA samples and is recommended if you need to retain biological activity. Gels can be cast and run in standard TAE or TBE Buffer Systems or MOPS Buffer. If there is a significant amount of secondary structure, another sample denaturation method should be chosen. Materials Water bath set to 70 C Ice Reagents AccuGENE Molecular Biology Water (RNase-free) Deionized formamide AccuGENE 10X MOPS Buffer (200 mm MOPS, ph 7.0, 50 mm sodium acetate, 10 mm EDTA, 10 mm EGTA), ph 7.0

141 Section VIII: Separation of RNA in Agarose Gels Preparation of RNA Samples continued Formamide denaturation of RNA samples 1. Bring the RNA volume up to 8 μl with RNase-free water. 2. Add 2 μl of 10X MOPS Buffer. 3. Add 9 μl of deionized formamide. 4. Mix thoroughly. 5. Heat at 70 C for 10 minutes. 6. Chill on ice for at least 1 minute before loading. Formaldehyde denaturation Formaldehyde denaturation is suitable when samples are to be recovered. It is necessary to ensure that the formaldehyde is fully removed from the recovered RNA prior to subsequent studies. Some enzymatic reactions, such as in vitro transcription, may be problematic even after complete removal of the formaldehyde. Materials Water bath set to 70 C Ice Reagents AccuGENE Molecular Biology Water (RNase-free) AccuGENE 10X MOPS Buffer (200 mm MOPS, ph 7.0, 50 mm sodium acetate, 10 mm EDTA, 10 mm EGTA), ph % (v/v) formaldehyde Deionized formamide Caution: Materials and methods shown here present hazards to the user and the environment. Refer to the safety information on page 210 before beginning these procedures. Formaldehyde denaturation of RNA samples 1. Bring the RNA volume up to 6 μl with RNase-free water. 2. Add 2 μl of 10X MOPS Buffer. 3. Add 2 μl of 37% formaldehyde. 4. Add 9 μl deionized formamide. 5. Mix thoroughly. 6. Heat at 70 C for 10 minutes. Chill on ice for at least 1 minute before loading. Glyoxal denaturation Glyoxal is a very efficient denaturant, but should not be used if samples are to be recovered. Glyoxal denatures RNA by introducing an additional ring into the guanosine residues, thus interfering with G-C base pairing. Glyoxal denaturation would be the recommended procedure for Northern blotting. Typically a phosphate electrophoresis buffer is recommended with recirculation to prevent formation of a ph gradient. Alternatively, a 10 mm PIPES, 30 mm bis-tris buffer, or a 20 mm MOPS, 5 mm sodium acetate, 1 mm EDTA, 1 mm EGTA Buffer systems can also be used without recirculation. Materials Water bath set to 50 C Ice Reagents AccuGENE Molecular Biology Water (RNase-free) DMSO 100 mm Sodium phosphate, ph 7.0 Mix 5.77 ml of 1 M Na 2HPO 4 with 4.23 ml of 1 M NaH 2 PO Adjust volume to 100 ml with RNase-free water 6 M Glyoxal, 40% (v/v) solution, deionized immediately before use Pass solution through a small column of mixed-bed ion exchange resin until the ph is >5.0; large volumes can be deionized then stored frozen in aliquots at 20 C Caution: Materials and methods shown here present hazards to the user and the environment. Refer to the safety information on page 210 before beginning these procedures. Glyoxal denaturation of RNA samples 1. Bring the RNA volume up to 11 μl with RNasefree water 2. Add 4.5 μl of 100 mm sodium phosphate. 3. Add 22.5 μl of DMSO 4. Add 6.6 μl of deionized glyoxal. 5. Mix thoroughly 6. Heat at 50 C for 1 hour. Chill on ice for at least 1 minute before loading Separation of RNA in Agarose Gels 133

142 Section VIII: Separation of RNA in Agarose Gels Buffers for RNA Electrophoresis The two commonly used buffer systems for RNA electrophoresis are a phosphate buffer for glyoxal/dmso denatured RNA and a MOPS Buffer for formaldehyde or formamide denatured RNA. These buffers are very low in ionic strength. During electrophoresis, a ph gradient may be generated along the length of the gel, resulting in the hydrolysis (melting) of the agarose gel. This problem can be avoided by recirculating the buffer. For glyoxaldenatured gels, if the ph of the buffer rises above ph 8.0, the glyoxal will dissociate from the RNA, causing the RNA to renature and migrate in an unpredictable manner. Buffer preparation NOTE: Use RNase-free chemicals, water and containers 100 mm Sodium Phosphate Buffer, ph 7.0 (Glyoxal/DMSO denatured RNA) g/l 1 M Na 2 HPO ml 1 M NaH 2 PO ml Adjust volume to 1 liter with RNase-free water Adjust volumes accordingly to prepare more buffer 10X MOPS Buffer (Formaldehyde or Formamide denatured RNA) g/l 200 mm MOPS (free acid) g 50 mm sodium acetate 6.80 g 2 O 3.72 g 10 mm EGTA (free acid) 3.80 g Mix with 850 ml of distilled water Adjust ph to 7.0 with 10 M NaOH Adjust volume to 1 liter with RNase-free water Filter through a 0.2 μm nitrocellulose filter and store in the dark Caution: NaOH is s corrosive material. Use safety glasses and gloves to protect from burns. Separation of RNA in Agarose Gels Section VIII: Separation of RNA in Agarose Gels Electrophoresis of RNA The choice of an agarose free of RNase contamination is of major importance. Lonza offers a variety of agarose products for RNA electrophoresis including The FlashGel System for RNA, Reliant and Latitude Precast RNA Gels, Lonza agarose, AccuGENE 10X MOPS Buffer, RNA Marker, GelStar and SYBR Green Nucleic Acid Gel Stains. General guidelines Northern blotting requires a standard melting temperature agarose such as SeaKem LE or NuSieve 3:1 Agarose or Reliant or Latitude Precast RNA Gels If samples are to be recovered, a low melting temperature agarose can be used such as NuSieve GTG or SeaPlaque GTG Agarose A % gel made with SeaKem GTG or SeaKem Gold Agarose or FlashGel System, Reliant or Latitude Precast RNA Gels will work for RNA molecules of ,000 nucleotides 134

143 Section VIII: Separation of RNA in Agarose Gels Electrophoresis of RNA continued For RNA smaller than 500 nucleotides, use a 3 or 4% NuSieve 3:1 or MetaPhor Agarose Gel For RNA larger than 10,000 nucleotides, SeaKem Gold Agarose and FlashGel System, Reliant or Latitude Precast RNA Gels will be a better choice for tighter bands and better resolution If a low melting temperature agarose is required, a 1.5 or 2.0% SeaPlaque GTG Gel can be used for separation of RNA from ,000 nucleotides, while a 3.0% or 4.0% NuSieve GTG Gel should be used for fine resolution of RNA smaller than 500 nucleotides; NuSieve GTG Agarose is not recommended for formaldehyde/mops gels Agarose selection guide for RNA electrophoresis Fragments <500 bases Fragments between ,000 bases Fragments >10,000 bases Dye mobilities for RNA gels The following table is a migration table of single-stranded RNA in relation to bromophenol blue (BPB) and xylene cyanol (XC) in formaldehyde or glyoxal agarose gels. Formaldehyde gels Glyoxal gels Agarose (% w/v) XC BPB XC BPB SeaKem Gold Agarose 1.0 6, , , , , , SeaKem GTG and SeaKem LE Agarose 1.0 4, , , , , SeaPlaque and SeaPlaque GTG Agarose 1.0 2, , , , NuSieve 3:1 Agarose , NuSieve GTG and MetaPhor Agarose 2.0 Not applicable 1, Not applicable Not applicable Separation Northern Blotting Preparative NuSieve 3:1 Agarose NuSieve 3:1 Agarose NuSieve GTG Agarose MetaPhor Agarose SeaKem LE Agarose SeaKem LE Agarose SeaPlaque GTG Agarose SeaKem GTG Agarose SeaKem GTG Agarose SeaKem Gold Agarose SeaKem Gold Agarose SeaPlaque GTG Agarose Separation of RNA in Agarose Gels 135

144 Section VIII: Separation of RNA in Agarose Gels Electrophoresis of RNA continued Separation of RNA in Agarose Gels Electrophoresis of gels containing formaldehyde A formaldehyde denaturant with a MOPS buffer is the most commonly used system for RNA electrophoresis. Care should be taken when handling gels containing formaldehyde. These gels are less rigid than other agarose gels. NuSieve GTG Agarose is not compatible with the MOPS buffering system. Formaldehyde electrophoresis of RNA NOTE: Do not exceed 20 μg of RNA per lane as larger amounts can result in loss of resolution. 1. For a 1% gel, dissolve 1.0 g of agarose in 72 ml of water. Adjust the amounts for different percent gels 2. Cool agarose to 60 C in hot water bath 3. Place in fume hood 4. Immediately add 10 ml of prewarmed 10X MOPS Buffer 5. Add 5.5 ml of prewarmed 37% formaldehyde 6. Cast gel in a fume hood 7. Denature the RNA sample following one of the methods previously described 8. Add 2 μl of formaldehyde loading buffer per 20 μl of sample 9. Mix thoroughly 10. Remove the gel comb 11. Place the gel in the electrophoresis chamber 12. Cover surface of the gel to a depth of 1 mm with 1X MOPS Buffer 13. Load the samples 14. Electrophorese at a maximum of 5 V/cm (interelectrode distance) until the bromophenol blue has traveled at least 80% of the way through the gel (see mobility table on page 135) Materials Water bath set to 60 C Fume hood Accessories to cast an agarose gel Electrophoresis chamber and power supply Flask or beaker Reagents AccuGENE 10X MOPS Buffer prewarmed to 60ºC 1X MOPS Buffer 37% (v/v), formaldehyde prewarmed to 60 C Formaldehyde Loading Buffer (1 mm EDTA, ph 8.0, 0.4% bromophenol blue and xylene cyanol, 50% glycerol) Distilled water Caution: Materials and methods shown here present hazards to the user and the environment. Refer to the safety information on page 210 before beginning these procedures. 136

145 Section VIII: Separation of RNA in Agarose Gels Electrophoresis of RNA continued Electrophoresis of gels containing glyoxal/dmso These gels should be run slower than formaldehyde gels with buffer recirculation to avoid the formation of a ph gradient. Glyoxylated RNA will give sharper bands than formaldehyde-treated RNA. Glyoxal/DMSO electrophoresis of RNA 1. For a 1% gel, dissolve 1.0 g of agarose in 100 ml of 10 mm sodium phosphate, ph 7.0. Adjust amounts for different percent gels. 2. Cool agarose to 60 C in hot water bath. 3. Cast the gels to a thickness that will accommodate a loading volume of 60 μl. 4. Remove the comb. 5. Place the gel in the electrophoresis chamber. 6. Cover gel with 10 mm sodium phosphate buffer to a depth of 1 mm. 7. Add 12 μl of glyoxal loading buffer per 45 μl of sample. 8. Mix thoroughly. 9. Load 0.5 μg μg of RNA per lane. 10. Electrophorese at 4 V/cm (interelectrode distance) while the buffer is recirculated. If no recirculation apparatus is available, pause electrophoresis every 30 minutes and remix the buffer. 11. Electrophorese until the bromophenol blue has traveled at least 80% of the way through the gel (see mobility table on page 135). Materials Water bath set to 60 C Fume hood Accessories to cast an agarose gel Electrophoresis chamber and power supply Flask or beaker Recirculating unit Reagents 100 mm sodium phosphate ph 7.0 Glyoxal loading buffer (10 mm sodium phosphate, ph 7.0, 0.25% bromophenol blue and xylene cyanol, 50% glycerol) Caution: Materials and methods shown here present hazards to the user and the environment. Refer to the safety information on page 210 before beginning these procedures. Separation of RNA in Agarose Gels 137

146 Section VIII: Separation of RNA in Agarose Gels Detection of RNA with GelStar or SYBR Green II Gel Stains Introduction Detection of RNA in agarose gels varies depending on the denaturant, stain and photographic conditions used. Lonza offers two highly sensitive stains, GelStar and SYBR Green II Gel Stains, for the detection of RNA in agarose gels. These stains exhibit higher RNA detection sensitivity than ethidium bromide, allowing you to load less RNA sample on your gel. Unlike ethidium bromide, GelStar and SYBR Green II Stain only #uoresce upon binding to the nucleic acid resulting in lower background #uorescence which is particularly useful when including the stain in the gel for glyoxal denatured samples or when concentrations of formaldehyde in the gel exceed 2 M. The chart below shows the detection sensitivity of various dyes for RNA using in-gel or post-staining techniques. Stain Method Native RNA Glyoxal RNA GelStar Stain in-gel 10 ng 10 ng Ethidium bromide, no destain in-gel 30 ng 150 ng Ethidium bromide, with destain in-gel 30 ng 50 ng SYBR Green II Stain post stain 15 ng 50 ng Limits are based on optimal detection methods for each stain. Samples detected with SYBR Green Stain were post-stained, samples detected with ethidium bromide or GelStar Stain were detected by in-gel staining (poststained gels showed similar results). A 2X working solution of GelStar or SYBR Green Stain should be prepared just prior to use from the 10,000X stock solution by diluting in 1X MOPS Buffer or a ph 7.5 to 8.5 buffer (e.g., TAE, TBE or TE). For nondenaturing or denaturing polyacrylamide gels, dilute GelStar or SYBR Gel Stain 1:10,000X in 1X electrophoresis buffer. Agarose gels should be cast no thicker than 4 mm. As gel thickness increases, diffusion of the stain into the gel is decreased, lowering the efficiency of RNA detection. Optimal sensitivity for GelStar and SYBR Green Stains is obtained by using the appropriate photographic filters for each stain. GelStar Stain: Wratten or Tiffen #9 Filter SYBR Green Stains: Wratten or Tiffen #15 Filter We do not recommend photographing gels with a 254 nm transilluminator. An outline of the UV light source may appear in photographs. A filter that will allow a 525 nm transmission and exclude infrared light is required. Separation of RNA in Agarose Gels FlashGel System (see page 25), uses a similar stain system and is capable of RNA detection <10ng. Tips for staining gels with GelStar or SYBR Green II Gel Stains Follow the guidelines below to increase the detection sensitivity of GelStar or SYBR Green II Gel Stains. New clear polypropylene containers (e.g., Rubbermaid Recycling #5 Plastics) should be obtained for use with GelStar and SYBR Green Stains. When stored in the dark in polypropylene containers, the diluted stain can be used for up to 24 hours and will stain 2 to 3 gels with little decrease in sensitivity. The containers should be rinsed with distilled water (do not use detergents) after each use and dedicated to GelStar or SYBR Green Stain use only These stains bind to glass and some non-polypropylene (polystyrene) plastics, resulting in reduced or no signal from the nucleic acid 138

147 Section VIII: Separation of RNA in Agarose Gels Detection of RNA with GelStar or SYBR Green II Gel Stains Continued Procedure for staining RNA with GelStar or SYBR Green II Gel Stains For optimal resolution, sharpest bands and lowest background, stain the gel with GelStar or SYBR Green II Gel Stain following electrophoresis. The photographs below demonstrate the detection sensitivity of various stains with different sample denaturants. Follow the steps below to stain RNA after electrophoresis 1. Remove the concentrated stock solution of the stain from the freezer and allow the solution to thaw at room temperature. 2. Spin the solution in a microcentrifuge to collect the dye at the bottom of the tube. 3. Dilute the 10,000X concentrate to a 2X working solution (2 μl/10 ml) in 1X MOPS Buffer, in a clear plastic polypropylene container. Prepare enough staining solution to just cover the top of the gel. 4. Remove the gel from the electrophoresis chamber. 5. Place the gel in staining solution. 6. Gently agitate the gel at room temperature. 7. Stain the gel for 60 minutes. 8. Remove the gel from the staining solution and view with a transilluminator, CCD camera or Dark Reader Transilluminator. NOTE: Gels stained with GelStar or SYBR Green II Gel Stains do not require destaining. The dyes fluorescence yield is much greater when bound to RNA than when in solution. Materials Clear polypropylene container (e.g., Rubbermaid Recycling #5 Plastics) GelStar Photographic Filter (Wratten #9 equivalent) or SYBR Green Photographic Filter (Wratten #15 equivalent) Microcentrifuge UV transilluminator Dark Reader Transilluminator (Clare Chemical Research, Inc.) or CCD imaging system Reagents 1X MOPS Buffer GelStar or SYBR Green II Gel Stain stock solution Caution: Materials and methods shown here present hazards to the user and the environment. Refer to the safety information on page 210 before beginning these procedures. Ethidium bromide GelStar Stain SYBR Green I Stain SYBR Green II Stain A B C A B C A B C A B C Separation of RNA in Agarose Gels 139

148 Section VIII: Separation of RNA in Agarose Gels Detection of RNA with GelStar or SYBR Green II Gel Stains continued Follow this procedure when including GelStar Nucleic Acid Gel Stain in the agarose gel NOTE: Unlike ethidium bromide, GelStar Nucleic Acid Gel Stain does not interact with glyoxal. 1. Remove the concentrated stock solution of GelStar Stain from the freezer and allow the solution to thaw at room temperature. 2. Spin the solution in a microcentrifuge tube. 3. Prepare the agarose solution. 4. Once the agarose solution has cooled to 70ºC, add the stain by diluting the stock 1:5,000 into the gel solution prior to pouring the gel (2 μl per 10 ml). 5. Slowly swirl the solution. 6. Pour the gel into the casting tray. 7. Load samples onto the gel. 8. Run the gel. 9. Remove the gel from the electrophoresis chamber. 10. View with a 300 nm UV Transilluminator, CCD camera or Clare Chemical s Dark Reader Transilluminator. NOTE: GelStar Stained Gels do not require destaining. The dye s fluorescence yield is much greater when bound to RNA than when in solution. GelStar SYBR Green I Stain SYBR Green II Stain Stain Stain A B C A B C A B C Samples of E. coli total RNA were denatured using the following denaturants: Lane A: Formaldehyde/Formamide; Lane B: Formamide; Lane C: Glyoxal. Samples were loaded at 2 mg/lane for the formaldehyde/ formamide and formamide only denatured samples, and 4 mg/lane for the glyoxal denatured samples. Reliant RNA Precast Agarose Gels were run at 7 V/cm for 40 minutes in 1X MOPS Buffer (prepared from AccuGENE 10X MOPS Buffer) and post-stained using GelStar or SYBR Green II Stain or ethidium bromide. The left series of photographs were taken using a Polaroid Camera, SYBR Green Photographic Filter on a UV light box. The right series of photographs were taken using the SYBR Green Photographic Filter on the Clare Chemical s Dark Reader Transilluminator. Separation of RNA in Agarose Gels 140

149 Section VIII: Separation of RNA in Agarose Gels Detection of RNA with GelStar or SYBR Green II Gel Stains Continued Visualization by photography Gels stained with GelStar and SYBR Green II Gel Stains exhibit negligible background uorescence, allowing long film exposures when detecting small amounts of RNA. Use the appropriate photographic filter for the stain you are using (see page 138). The table below provides suggested film types and photographic conditions Polaroid Film f-stop Exposure time Type 57 or seconds Type seconds Visualization by image capture system GelStar and SYBR Green II Nucleic Acid Gel Stains are compatible with most CCD and video imaging systems. Due to variations in the filters for these systems, you may need to purchase a new filter. Lonza does not sell filters for this type of camera. Contact your systems manufacturer and using the excitation and emission information listed, they can guide you to an appropriate filter. Stain Emission (nm) Excitation (nm) GelStar Stain SYBR Green I Stain SYBR Green II Stain Materials Staining vessel larger than the gel UV transillu minator, Dark Reader Transillu minator (Clare Chemical Research, Inc.) or CCD imaging system Magnetic stir plate Magnetic stir bar Reagents Ethidium bromide stock solution (10 mg/ml) (1.0 g Ethidium bromide,100 ml distilled water, stir on magnetic stirrer for several hours, transfer the solution to a dark bottle, store at room temperature) Distilled water 0.1 M ammonium acetate Caution: Materials and methods shown here present hazards to the user and the environment. Refer to the safety information on page 210 before beginning these procedures. Application notes The uorescent characteristics of GelStar and SYBR Green II Gel Stains make them compatible with argon ion lasers Gels previously stained with ethidium bromide can subsequently be stained with GelStar or SYBR Green II Gel Stain following the standard protocol for poststaining. There will be some decrease in sensitivity when compared to a gel stained with only GelStar or SYBR Green II Gel Stain We recommend the addition of 0.1% to 0.3% SDS in the prehybridization and hybridization solutions when performing Northern blots on gels stained with these dyes Separation of RNA in Agarose Gels 141

150 Section VIII: Separation of RNA in Agarose Gels Detection of RNA with Ethidium Bromide Separation of RNA in Agarose Gels Introduction Ethidium bromide does not stain RNA as efficiently as it does DNA, so be certain that sufficient RNA is loaded to see the band of interest. Although as little as 10 ng - 20 ng of DNA can usually be visualized by ethidium bromide uorescence, as much as 10-fold more RNA may be needed for good visualization. Visualization of poorly staining RNA is made even more difficult by the higher background uorescence of RNA gels. In formaldehyde gels, background uorescence can be minimized by dropping the formaldehyde concentration in the gel from 2.2 M to 0.66 M. Staining with ethidium bromide is not recommended when performing Northern blot analysis onto nylon membranes. Follow the steps below to stain RNA after electrophoresis 1. Prepare enough working solution of ethidium bromide (0.5 mg/ml of ethidium bromide in 0.1 M ammonium acetate) to cover the surface of the agarose gel. 2. Remove the gel from the electrophoresis chamber. 3. Submerge the gel for minutes in the ethidium bromide solution. 4. Remove the gel from the solution. 5. Submerge the gel for 60 minutes in a new container filled with distilled water. NOTE: For gel concentrations of 4% or greater, these times may need to be doubled. If after destaining the background is still too high, continue to destain. 6. Repeat in fresh distilled water. 7. Gels can be viewed with a UV light transilluminator, Dark Reader Transilluminator (Clare Chenical Research, Inc.) or CCD imaging system. Follow the steps below when including ethidium bromide in the agarose gel 1. Prepare agarose solution. 2. While the agarose solution is cooling, add ethidium bromide to a final concentration of 0.5 μg/ml to the solution. 3. Slowly swirl the solution. 4. Pour the gel into the casting tray. 5. Add ethidium bromide to the running buffer to a final concentration of 0.5 μg/ml. 6. Load and run the gel. 7. Destain the gel by submerging the gel in distilled water for 60 minutes. 8. Repeat in fresh distilled water. 9. Gels can be viewed with a UV light transilluminator, Dark Reader Transilluminator (Clare Chemical Research, Inc.) or CCD imaging system. Decontamination of GelStar and SYBR Green Gel Stains and ethidium bromide solutions Staining solutions should be disposed of by passing through activated charcoal followed by incineration of the charcoal. For absorption on activated charcoal, consult Sambrook, et al., pp , (1989). Follow state and local guidelines for decontamination and disposal of Nucleic Acid Staining Solutions. 142

151 Section VIII: Separation of RNA in Agarose Gels Northern Blotting Recommended agaroses for Northern blotting When transferring RNA from an agarose gel to a membrane, a standard melting temperature agarose should be used. Standard melting temperature agaroses have higher gel strength than low melting temperature agaroses and do not fracture during transfer. The table below is a list of Lonza agaroses and precast gels that are recommended for blotting. Agarose Size Range (bases) NuSieve 3:1 Agarose <500 SeaKem LE Agarose ,000 SeaKem GTG Agarose ,000 SeaKem Gold Agarose ,000 Reliant & Latitude RNA Gels 25-10,000 Tips for agarose gel preparation Use the lowest agarose concentration required to resolve your fragments If staining with ethidium bromide prior to transfer, thoroughly destain the gel with distilled water Avoid casting thick gels (>4 mm) unless absolutely necessary. Thick gels not only require longer electrophoretic times, but may interfere with the free transfer of nucleic acids to the hybridization membrane Do not use more than a gm weight on top of the stack. Excess weight can compress the gel. This will increase the agarose concentration and decrease the pore size, which can inhibit movement of the buffer and RNA Choosing the appropriate membrane When transferring RNA, nylon membranes have several advantages over nitrocellulose: Nylon Nitrocellulose Uncharged/charged Supported/unsupported High strength Good strength* Good for reprobing Poor for reprobing UV crosslinking Baking in vacuum oven at 80ºC 50 nucleotides** 500 nucleotides** *Good for supported nitrocellulose, poor for unsupported nitrocellulose. **Lower size limit for efficient nucleic acid retention. Choosing a blotting method Consider the following when choosing a blotting method: Gel concentration Fragment size Speed and transfer efficiency Capillary transfer Traditional passive capillary transfer uses paper towels to draw the transfer buffer from a reservoir through the gel. Passive capillary transfer takes hours to complete. The most common complaints are poor transfer efficiency for larger RNA molecules and long transfer time. Vacuum transfer Vacuum transfer systems use negative pressure to pull the transfer buffer through the gel along with the nucleic acid. These systems take approximately minutes to complete. Vacuum blotting can be useful for high concentration gels where compression of the gel is a concern. Electroblotting Electroblotting is most often used for polyacrylamide gels. A voltage gradient pulls the sample out of the gel and onto a membrane. Semi-dry electroblotting requires minimal buffer with a low voltage and current. Care should be taken so smaller fragments are not pulled completely through the membrane. Separation of RNA in Agarose Gels 143

152 Section VIII: Separation of RNA in Agarose Gels Northern Blotting continued Separation of RNA in Agarose Gels General guidelines for Northern blotting It is important to minimize RNase activity by following certain precautions. There are several agents on the market that effectively remove RNase s or consult Sambrook, et al Wear gloves throughout the procedure. RNAs are not safe from nuclease degradation until they have been immobilized on the membrane Cut or mark membrane for orientation. Do this before wetting the membrane The blotting membrane should be in contact with the underside of the gel. Since nucleic acids will concentrate near the bottom of the gel, there is less distance for them to travel during the transfer Cut all papers and membranes to the correct size, such that, the only thing pulling the transfer buffer is the buffer solution Avoid bubbles, ensure that there is even contact between all the layers of the blotting system Incorrect denaturation of probe can cause poor transfer results Glyoxal gels can be transferred immediately after electrophoresis. The glyoxal will be removed in the post transfer, prehybridization wash Destain gels that contain formaldehyde or ethidium bromide to avoid sample loss and inefficient transfer efficiency NOTE: Soak gel 3 times for 5-10 minutes each in either 1X MOPS, transfer buffer, or sterile water. This will remove most of the formaldehyde and excess background uorescence seen with ethidium bromide. Complete destaining usually takes 2 hours or longer. Lonza s Reliant and Latitude Precast RNA Gels do not contain any denaturants or stains and do not require destain prior to transfer. Materials Nitrocellulose or nylon membrane 2-3 glass dishes larger than gel Paper towels Whatman 3MM Chromatography Paper Glass or plastic pipette Flat ended forceps Glass plate to serve as a platform for the gel Plastic wrap <500.0 g weight Orbital or rocking platform shaker Reagents AccuGENE Molecular Biology Water AccuGENE 20X SSPE or AccuGENE MOPS Buffer Procedure for RNA transfer by passive capillary electrophoresis transfer buffer The concentration of the transfer buffer will vary depending on the method and type of membrane used. The capillary method outlined here uses a nylon membrane and 5X SSPE Buffer. 20X SSPE (3 M NaCl, 0.2 M NaH 2 PO O) g NaCl 27.6 g NaH 2 PO 4 2 O 2 O Adjust to ph 7.4 with 10 N NaOH To 1 liter with distilled water 144

153 Section VIII: Separation of RNA in Agarose Gels Northern Blotting Continued Follow the steps below for gel preparation and setting up the transfer 1. Cast a standard melting temperature agarose gel no thicker than 4 mm. 2. Electrophorese RNA following standard protocols. 3. For formaldehyde gels, follow the steps below prior to transfer. For glyoxal gels, proceed to step Soak the gel in an excess of 1X MOPS Buffer, distilled water or 5X SSPE for 10 minutes. 5. Repeat three times with new wash solution each time. 6. Float membrane in RNase-free water for 5 minutes. 7. Equilibrate membrane in 5X SSPE for 5 minutes. The membrane may remain in the transfer buffer until it is used. 8. Set up transfer using either an upward capillary transfer set up or downward capillary transfer set up. 9. Allow the transfer to proceed as follows: Transfer set-up Time Upward hours Downward 1-4 hours depending on gel thickness and concentration Turboblotter System 1-3 hours 10. Remove the paper toweling or chromatography paper. 11. Remove the gel and membrane together. 12. Mark the positions of the wells on the membrane with a pencil for orientation then remove the gel from the transfer setup. 13. Rinse the membrane for seconds in transfer buffer. 14. Place the membrane on a sheet of Whatman 3MM Chromatography Paper. 15. Treat membrane as described in Immobilizing RNA on a membrane, next section. Materials Membrane Plastic wrap UV light source Vacuum dryer set to 80 C or hot oven at 65 C. Reagents AccuGENE 1 M Tris ph 8.0 RNase-free water AccuGENE Molecular Biology Water or AccuGENE 10X Mops Buffer AccuGENE 20X SSPE Immobilizing RNA on a membrane The methods and procedures for immobilizing RNA on the membrane are essentially the same as they are for DNA. If samples have been denatured under the formaldehyde system, immobilization can take place immediately after transfer without any pretreatment steps. The formaldehyde has been removed from the system prior to transfer during the gel destaining steps. If the glyoxal denaturing system was used, the glyoxal must now be removed from the filter. Follow one of the procedures below to remove glyoxal from the membrane. Option 1 1. Air dry the membrane. 2. Bake the membrane as follows: Membrane Type Temperature Time Nitrocellulose 80 C 2 hours under vacuum Nylon 65 C hours 3. Immerse the filter in 200 ml of preheated 20 mm Tris, ph Cool to room temperature. Separation of RNA in Agarose Gels References Ausubel, F.E., et al., Current Protocols in Molecular Biology, J. Wiley and Sons, Inc.,1998. Farrell, R.E. Jr., RNA Methodologies: A Laboratory Guide for Isolation and Characterization, 2nd Edition, Academic Press, Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press, White, H.W., et al., BioTechniques 26: , Grundemann, D. & Koepsell, H., Anal. Biochem. 216: , Option 2 1. Immobilize RNA on membrane either by UV irradiation or baking. 2. Wash membrane 65 C for 15 minutes in 20 mm Tris, ph

154 Section VIII: Separation of RNA in Agarose Gels FlashGel System for RNA Analysis The FlashGel System completes RNA analysis in less than 30 minutes and requires one-fifth the amount of total RNA for detection. The FlashGel System is recommended for verification and analysis of total RNA (Figure 1), quick checks of native RNA (Figure 3) and checks for RNA degradation (Figure 2) Rapid RNA analysis Separation of RNA in Agarose Gels The FlashGel System for RNA separates up to 34 samples of RNA in 8 minutes or less. RNA samples are visible on the FlashGel Dock for up to 4 minutes, after which they fade and then reappear with increasing intensity following a minute post-run hold. Full analysis is complete in less than 30 minutes, compared to the 1-3 hours required for typical agarose gels. Figure 1 demonstrates analysis time of the FlashGel System compared to other agarose gel methods for RNA. Sensitive detection RNA quantities <10 ng per band are clearly detected on the FlashGel System for RNA. Some RNA preparations deliver such low yields that there may not be the surplus RNA needed to assess integrity. The FlashGel System for RNA uses a stain that reduces the amount of required RNA by a factor of five or greater compared to ethidium bromide. Figure 1 illustrates detection sensitivity of the FlashGel System for RNA compared to other agarose gel and staining methods. Clean, enclosed system The FlashGel System for RNA eliminates the hazards and tedium associated with RNA gel preparation. The cassettes fully enclose the gel and running buffer, eliminating operator exposure to hazardous reagents, and protecting samples from contaminating RNases. The cassettes contain a 1.2% agarose and buffer blend which are selected for purity, manufactured in a dedicated clean room, and guaranteed RNase-free. The FlashGel Dock does not come in contact with samples or gels, so it is not necessary to dedicate a unit for RNA work. A A B C D Panel Gel Sample preparation Run conditions Total time B C D FlashGel System for RNA Reliant 1.25% MOPS Gel (Cat. #54922) Reliant 1.25% MOPS Gel Hand-cast 1% SeaKem LE Agarose Gel (Cat. #50002) Formaldehyde Sample Buffer (Cat.# 50571) Samples denatured 5 Min. at 65 C. 10μg/ml EtBr added to Formaldehyde Sample Buffer. Samples denatured 5 Min. at 65 C. 8 min run at 225 V. Cassette held at ambient temp for 20 min prior to imaging. 60 min run at 5 V/cm. 60 min post-stain with GelStar Stain (Cat. #50535) 1:5000 dilution 60 min run at 5 V/cm. 60 min EtBr post-stain and 60 min de-stain. Denaturing gel prepared at 2.2 M Formaldehyde in 1X MOPS buffer (Cat. #50876). 60 min run at 6 V/cm. Figure 11. S Sample l f RNA of RNA M k Marker (C #50575) (Cat. # 50575) l 1 & lanes 2 d1 & 2, Tand l RNA E. coli (A Total bi ) RNA (Ambion) Lanes 3 & 4 contain 50 ng (lanes 1 & 3 or 250 ng (lanes 2 & 4) of RNA per 5μl load min 120 min 108 min 120 min Figure 2. Checking Sample Quality with the FlashGel System for RNA Sample degradation is clearly visible on the FlashGel System for RNA. 8 min run at 225 V, followed by 20 min hold prior to imaging. Lane 1: RNA Marker (Lonza); Lane 2: 250 ng E.coli Total RNA (Ambion); Lanes 3-7: 250 ng E.coli Total RNA incubated with increasing levels of RNase A. Intact, denatured RNA shows sharp, clear bands on the FlashGel System. Partially degraded RNA has a smeared appearance, and completely degraded RNA appears as a low molecular weight smear. 146

155 Section VIII: Separations of RNA in Agarose Gels FlashGel System for RNA Analysis continued Simple Protocol Procedure 1. Insert cassette into FlashGel Dock. 2. Pre-load wells with RNase-free Water (Cat. #51200). 3. Load samples Plug in and turn on light and power (225 V). 5. Run for 8 minutes Turn off power and hold for minutes or until desired RNA bands are visible on the gel. 7. Photograph as usual Formaldehyde Loading Buffer and RNA Markers are recommended for best performance. Denatured RNA samples will migrate according to their true size on FlashGel RNA Cassettes. 2. The FlashGel DNA Marker may be used to monitor separation since it will immediately be visible on the dock in real time and be visible for the full length of the run - stop the run when the 100 bp band reaches the end of the gel. The visible blue xylene cyanol band may also be used to track migration - stop the run when the blue band has migrated approximately 1/3 the distance of the gel. 3. RNA bands are visible for up to 4 minutes in realtime runs (see Figure 3). If desired bands are seen within that time, then a minute hold time prior to imaging is not required. The longer the post-run incubation period (up to 20 minutes), the brighter the RNA bands become. Unlike typical agarose gels where samples diffuse immediately, RNA samples on FlashGel Cassettes remain intact for >2 hours. Caution: Materials and methods shown here present hazards to the user and the environment. Refer to the safety information on page 210 before beginning these procedures. Figure 3. Quick checks of Native RNA on the FlashGel System for RNA Native and formaldehyde denatured RNA samples were run at 225 V for 4 min. Native, nondenatured RNA samples do not require additional incubation time compared to formaldehyde denatured samples, and may be suitable for very fast checks of RNA integrity. After 20 additional minutes, the denatured samples would absorb stain and increase in visibility. Lane Sample Load volume Preparation RNA Marker (Lonza) E. coli Total RNA (Ambion) RNA Marker E. coli Total RNA 1.25 l 1.25 l 5 l 5 l Native samples prepared with FlashGel Loading Dye 5 RNA Marker 1.25 l Formaldehyde denaturation 6 E. coli Total RNA 1.25 l at 65ºC for 7 RNA Marker 5 l 5 minutes 8 E. coli Total RNA 5 l Some components and technology of the FlashGel System are sold under licensing agreements. The nucleic acid stain in this product is manufactured and sold under license from Molecular Probes, Inc., and the FlashGel Cassette is sold under license from Invitrogen IP Holdings, Inc, and is for use only in research applications or quality control, and is covered by pending and issued patents. The FlashGel Dock technology contains Clare Chemical Research, Inc. Dark Reader Transilluminator technology and is covered under US Patents 6,198,107; 6,512,236; and 6,914,250. The electrophoresis technology is licensed from Temple University and is covered under US Patent 6,905,585. Separation of RNA in Agarose Gels 147

156 148

157 Section IX: Special Applications in Agarose Gels In This Section Amplification of Plasmid cdna Libraries with SeaPrep Agarose 150 Preparing Agarose for use in Cell Culture Applications 152 References 154 Special Applications in Agarose Gels 149

158 Section IX: Special Applications in Agarose Gels Amplification of Plasmid cdna Libraries with SeaPrep Agarose Special Applications in Agarose Gels Introduction This section describes an amplification technique developed to address the common problem of disproportionate amplification of plasmids seen when expression cloning mrnas of very low abundance encoding cytokines, receptors, and cell surface molecules from plasmid cdna libraries derived from highly complex tissue sources. This amplification technique allows amplification of a plasmid cdna library in a representative fashion, decreasing the possibility that less abundant clones would vanish during the amplification due to differential rates of replication. The technique below describes a method where bacterial transformants are suspended, not plated, in low gelling temperature agarose. With this method, it has been found that, from generation to generation of amplification, the relative abundance of bacterial cells containing plasmids encoding selected hematopoietic factors varies less than two fold. Preparing the bacterial growth media plus agarose NOTE: Alternatively, prepared solutions of L Broth, LB Broth or Super Broth can also be used. 1. In a 2 liter Erlenmeyer flask, add the following reagents to 1 liter of double distilled water: 1a g bacto-tryptone 1b g bacto-yeast extract 1c g NaCl 2. Measure ph of solution. 3. If necessary, adjust to ph 7.0 using 1 N NaOH or 1 N HCl. 4. Add 3.0 g of SeaPrep Agarose to the solution. 5. Autoclave the solution on a liquid cycle for minutes. 6. Cool the solution to 40 C. 7. Store in a 37 C water bath until ready-for-use. 8. Prior to use add 2 ml of 50 mg/ml ampicillin. cdna/vector annealing reactions 1. Prepare vector using a tailing method. 2. Prepare and purify cdna following standard procedures. 3. Optimize the tailing of cdna inserts following standard procedures. 4. Scale up the appropriate cdna/vector-annealing reaction to generate up to 2.5 x 10 6 cfu/2000 ml. 5. Perform the appropriate number of standard bacterial transformations following standard procedures. 6. Pool all the transformations after the 37 C incubation. NOTE: General procedures for vector and cdna preparation, ligation and transformation can be found in Sambrook, J., et al. and Kriegler, M. Materials Autoclave 2 L Erlenmeyer μasks Water bath set to 37 C ph meter Sterile 50 ml polypropylene centrifuge tubes Ice water bath Sterile 500 ml centrifuge bottle Centrifuge Sterile Nunc Tubes 0.2 μm filter Reagents SeaPrep Agarose 2X L Broth (LB) 50 mg/ml ampicillin Double distilled water Bacto-tryptone Bacto-yeast extract NaCl 1 N NaOH or 1 N HCl ph standards LB-amp plates Filter sterilized glycerol 150

159 Section IX: Special Applications in Agarose Gels Amplification of Plasmid cdna Libraries with SeaPrep Agarose continued Plasmid cdna library amplification 1. Add ampicillin to a final concentration of 100 μg/ml (2.0 ml of 50 mg/ml stock solution) to the 2X LB-agarose solution. 2. Add transformants (1.25 x 10 6 cfu) to the LB-agarose solution. 3. Gently swirl to avoid foaming. 4. Aliquot the transformation mix into 25 ml aliquots in 50 ml polypropylene centrifuge tubes. 5. Place the tubes in an ice-water bath for minutes to allow the agarose gel to set. 6. Incubate overnight at 37 C undisturbed. 7. Plate 100 μl of the cell-agarose suspension directly on LB-amp plates for titer determination or prepare the suspension for library storage. Library Storage 1. Prepare 12.5% glycerol by diluting in double distilled water. 2. Filter solution through a 0.2 μm filter into a sterile container. 3. Pour the colony-containing gel into a sterile 500 ml centrifuge bottle. 4. Pellet the cells at 8,000 rpm for 20 minutes at room temperature. It is not necessary to melt the gel, the cells will pellet through the soft agarose. 5. Decant the media from the cell pellets. 6. Resuspend the cell pellets in 100 ml of 12.5% glycerol in 2X L broth. 7. Aliquot into sterile Nunc tubes. 8. Stores library at 70 C. Materials Scalpel or razor blade GelBond Film Glass plate or press board Clamps or elastic bands A forced hot-air oven Reagents SeaKem LE or GTG Agarose SeaKem Gold Agarose NuSieve 3:1 or NuSieve GTG Agarose MetaPhor Agarose References Kriegler, M., Gene Transfer and Expression: A Laboratory Manual, Stockton Press, Perez, C. and Kriegler, M., Resolutions, 6(4): 1-2, Ausubel, F.M., et al., Current Protocols in Molecular Biology, Wiley & Sons, Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor 151 Special Applications in Agarose Gels

160 Section IX: Special Applications in Agarose Gels Preparing Agarose for use in Cell Culture Applications Introduction Agarose has unique properties that make it especially useful in cell culture media applications. Agarose can form stable gels at concentrations as low as 0.3% (w/v), has very low ionic concentrations and is free of contaminating impurities that may affect the growth characteristics of cells in culture. Agarose could be used directly without supplementation for plaque assays ensuring high cell variability. The use of agarose is suggested for media where the absence of a nutrient is mandatory. Advantages Highly purified, eliminating contaminants Excellent optical transparency which enhances colony observation Gelling temperatures below 28 C allowing manipulation of cells in solution Can be used to produce a defined media system Applications Cell culture media Mammalian Plant Bacterial Viral Overlay assays Plaque hybridization assays Soft agarose hybridoma cloning Attachment dependent cell culture Colony lift assays Analysis of matrix and cell matrix interactions The concentration and type of agarose to use largely depends on your cell system, the application, and the lot specific gel strength of the agarose. There are three types of agarose which can be used for cell culture media, each having unique properties making one more suitable for a given application than another. SeaKem LE Agarose A standard melting and gelling temperature agarose which can be used as a solid medium to support cell growth by supplementation with growth factors and nutrients, top agar enriched with magnesium for baculovirus screening, substrate for bacterial growth and for colony lifts SeaPlaque Agarose A low gelling temperature agarose (28 C) that remains a liquid at 37 C allowing the manipulation of cells within the solution; SeaPlaque Agarose can be used as a semi-solid media for anchorage independent assays, plaque assays or overlays; SeaPlaque Agarose has also been found to be very effective as a medium for protoplast culture SeaPrep Agarose Unique ultra-low gelling temperature (15 C) and gel strength agarose (>75 g/cm 2 ); SeaPrep Agarose is ideal for hybridoma cloning; Cells can be recovered from the gel by increasing the temperature slightly, allowing transfer to a viable cell suspension for subsequent growth in liquid medium Special Applications in Agarose Gels 152

161 Section IX: Special Applications in Agarose Gels Preparing Agarose for use in Cell Culture Applications continued Suggested Agarose Products and Concentration Guidelines The table below provides general guidelines on agaroses and gel concentrations for given applications. General guidelines and specific information pertaining to a given cell type and application can be obtained from the current literature and Cell Biology: A Laboratory Manual. Agarose Preparation When preparing agarose for cell culture work, it is always best to prepare the agarose in water suitable for cell culture and separate from any growth media or nutrients. Agarose solutions and media solutions should be prepared at 2X concentrations (i.e., if desired final agarose concentration is 0.6%, prepare a 1.2% agarose solution), autoclaved separately, and aliquoted into useable aliquots. Application SeaKem LE Agarose SeaPlaque Agarose SeaPrep Agarose Mammalian cell culture 0.3% - 1% 0.25% - 1.5% Baculovirus screening 0.3% - 0.6% Bacterial culture 0.7% - 1.0% 1.0% Colony lifts 0.7% - 1.0% 1.0% Anchorage independent assays 0.25% - 0.5% Plaque assays 0.3% - 1% 0.50% - 1.5% Overlays 0.3% - 0.6% 0.3% - 1% Protoplast culture 0.35% - 0.7% Hybridoma cloning 0.8% - 1.5% Virus enumeration 1% Cell matrix interactions 1.0% - 3.0% Attachment dependent cell culture 0.6% In vitro cloning mammalian 0.5% - 2% 1% - 4% NOTES: Higher agarose concentration gels can affect and possibly restrict cell proliferation. Optimal gel concentrations should be determined for each culture system on a case-by-case basis. In some cases, such as hybridoma cloning using SeaPrep Agarose, it is advisable to sample different lots of agarose for the desirable gel strength qualities. Special Applications in Agarose Gels 153

162 Section IX: Special Applications in Agarose Gels Preparing the Agarose for Use in Cell Culture Applications continued Procedure for Autoclaving Agarose 1. Choose a flask that is 2-4 times the volume of the solution. 2. Add water to the flask. 3. Sprinkle in the pre-measured agarose powder at a 2X final agarose concentration. 4. Cover the flask with aluminum foil. 5. Place the flask in the autoclave. 6. Sterilize the agarose by autoclaving for 10 minutes at 15 lb/in 2. If using SeaPrep Agarose, autoclave for no longer than 5 minutes. NOTE: Agarose may lose gel strength when exposed to longer periods of time in the autoclave. 7. Once the agarose solution has cooled, aliquot into useable aliquots and store at 4 C prior to use. Materials Flask that is 2-4 times the volume of the solution Autoclave Aluminum foil Sterile asks or culture tubes Reagents Agarose powder Water suitable for cell culture General Procedure for Using Agarose in Culture Medium 1. Remelt the agarose by placing in a hot water bath or microwave. 2. Allow the agarose solution to cool to 37 C. 3. Prewarm the 2X media solution to 37 C. 4. Mix equal volumes of the sterile 2X agarose solution with sterile 2X media containing growth factors and nutrients. 5. Cast the agarose/media solution into plates or sterile culture tubes. 6. Allow the agarose solution to gel for 20 minutes if using as a feeder or overlay or maintain the solution at 37 C if using as a liquid culture. NOTE: A solution containing 1% SeaPlaque Agarose will stay liquid for approximately 18 hours. The amount of time an agarose solution will stay in a liquid state at 37 C, largely depends on the agarose concentration (increased agarose concentrations will decrease the time the solution stays in a liquid state), the age of the agarose and the particular lot of agarose you are using. When purchasing a new lot of agarose, we recommend you test this prior to culturing cells. Materials Microwave or hot water bath Reagents Gelled agarose solution 2X media solution Special Applications in Agarose Gels 154 References Borden, E.C., et al., Appl. Microbiol. 20: , Prescott, D.M., Methods in Cell Biology, 1st edition, Academic Press, Saunders, F.J.K. and Smith, J.D., Nature 227: , Mack, D.O., et al., BioTechniques, 17(5): , Buschmann, M.D., et al., J. of Orthopaedic Research, 10: , Calba, H., et al., Plant and Soil 178: 67-74, Shantze, K.A., et al., Cancer Research 45: , Celis, J.E., Cell Biology: A Laboratory Manual, 2nd edition, Academic Press, Bagasra, O., et al., Cancer Immunol Immunother, 20: 55-60, Civin, C.I., et al., J. Immunol. Methods, 61: 1-8, Buschmann, M.D., et al., J. of Cell Science, 108: , 1995.

163 Section X: Protein Separation in Polyacrylamide Gels Protein Separation in Polyacrylamide Gels In This Section Buffers for Protein Electrophoresis 156 Casting Polyacrylamide Gels 157 Loading and Running Proteins on Polyacrylamide Gels 159 Loading Buffers 160 Optimal Voltage, Running Times and Power Settings 161 ProSieve 50 Gel Solution 161 PAGEr Gold Precast Polyacrylamide Minigels 162 Chamber Modification Instructions 164 Detection of Proteins in Polyacrylamide Gels 166 SYPRO Protein Gel Stains 166 Coomassie Blue Stain 168 Silver Stain 169 References

164 Section X: Protein Separation in Polyacrylamide Gels Buffers for Protein Electrophoresis Protein Separation in Polyacrylamide Gels Overview Polyacrylamide gel electrophoresis (PAGE) is a powerful tool for separating and identifying mixtures of proteins and peptides. Several systems exist for performing PAGE and consideration should be given to which system best suits a given sample prior to running the samples. Gradient vs. homogeneous (straight percentage) gels Gradient gels are suitable for a wide range of size resolutions, and will result in tighter band separation than single concentration gels. A homogeneous, or single concentration gel is appropriate where the proteins of interest are known to be within a narrow size range. Discontinuous and continuous buffer systems A discontinuous buffer system utilizes a large-pore gel (the stacking gel) layered on top of a small pore gel (the resolving gel). In this system, the buffer used to prepare the gels is different from the buffer used in the tank. The different buffers create two ion fronts concentrating the proteins into a very tight zone. A continuous buffer system uses the same buffer in the gel and the tank. This system uses a single separating gel (no stacking gel) and has a single ion front. This system is less used than discontinuous systems, however, the separation of specific proteins and protein complexes that precipitate or aggregate may require the use of a continuous system. Buffers for Protein Electrophoresis The Laemmli Buffer System (Tris-Glycine) is a discontinuous buffer system, widely used for fine resolution of a broad molecular weight range of proteins. In this system, the gel is prepared with Tris-HCl Buffer and the Tris-glycine is used as the running buffer. In the Tris-Tricine Buffer System, tricine replaces glycine in the running buffer. The result is more efficient stacking and destacking, and higher resolution of proteins and peptides with lower molecular weights (under 10 kda - 15 kda). Buffer Preparation Tris-Glycine SDS Buffer, ph X Stock Solution g/l for 10X Stock Solution.25 M Tris base 30.3 g Tris Base 1.92 M Glycine g Glycine 1.0% SDS* 10.0 g SDS Adjust volume to 1 liter with distilled water (1X = 25 mm Tris base, 192 mm Glycine, 0.1% SDS*) *Omit SDS if running native proteins. Tris-Tricine SDS Buffer, ph X Stock Solution g/l for 10X Stock Solution 1 M Tris base g Tris base 1 M Tricine g Tricine 1.0% SDS* 10.0 g SDS Adjust volume to 1 liter with distilled water (1X = 100 mm Tris base, 100 mm Tricine, 0.1% SDS*) *Omit SDS if running native proteins. 156

165 Section X: Protein Separation in Polyacrylamide Gels Casting Polyacrylamide Gels Casting Polyacrylamide Gels Follow the steps below to cast a straight percentage polyacrylamide gel with a stacking gel. Alternatively, Lonza offers ready-to-use ProSieve 50 Gel Solution, or PAGEr Gold Precast Gels in a wide variety of concentrations and well formats. Casting the resolving gel 1. Assemble plates according to the manufacturer s instructions. 2. Place the specified quantity of the first four components from the table on page 175 into a side arm flask. 3. Mix gently by swirling. NOTE: Omit SDS if running a native gel. 4. Degas the solution for 15 minutes. A high concentration of APS (0.3%) can be used to speed polymerization and skip the degassing step. 5. Add the specified amounts of TEMED and 10% APS. 6. Mix gently by swirling. 7. Pour the resolving gel, leaving space for the stacking gel. 8. Gently overlay the acrylamide with water-saturated isobutanol. The overlay blocks oxygen from inhibiting polymerization of the resolving gel. 9. Allow the gel to polymerize for minutes. A very sharp liquid-gel interface will be visible when the gel has polymerized. 10. Pour off the overlay. 11. Rinse the top of the gel several times with water. 12. Blot any remaining water with a paper towel. Materials Vertical electrophoresis plate assembly with comb Sidearm ask assembly or filter unit for degassing solutions Paper towels Reagents 30% Acrylamide mix, 29% (w/v) Acrylamide, 1% (w/v) bisacrylamide, prepared in distilled water 1.5 M Tris Buffer ph % SDS 10% Ammonium Persulfate (APS) TEMED Distilled water Water saturated isobutanol Caution: Wear gloves, safety glasses and lab coats when handling acrylamide solutions. Protein Separation in Polyacrylamide Gels 157

166 Section X: Protein Separation in Polyacrylamide Gels Casting Polyacrylamide Gels continued Protein Separation in Polyacrylamide Gels Preparation of Tris-Glycine Resolving Gel Component volumes (ml) Solution components 10 ml 20 ml 50 ml 6% Distilled water % Acrylamide mix M Tris (ph 8.8) % SDS % Ammonium persulfate TEMED % Distilled water % Acrylamide mix M Tris (ph 8.8) % SDS % Ammonium persulfate TEMED % Distilled water % Acrylamide mix M Tris (ph 8.8) % SDS % Ammonium persulfate TEMED % Distilled water % Acrylamide mix M Tris (ph 8.8) % SDS % Ammonium persulfate TEMED % Distilled water % Acrylamide mix M Tris (ph 6.8) % SDS % Ammonium persulfate TEMED NOTE: Adjust proportionally based on the amount of gel needed. Preparation of a 5% stacking gel 1. Place the specified quantity of the first four components from the table below into a side arm flask. Component volumes (ml) Solution components 1 ml 5 ml 10 ml Distilled water % Acrylamide mix M Tris (ph 8.8) % SDS % Ammonium persulfate TEMED Mix gently by swirling. NOTE: Omit SDS if running a native gel. 3. Degas the solution for 15 minutes. 4. Add the specified amounts of TEMED and 10% APS. 5. Mix gently by swirling 6. Pour the stacking gel directly onto the resolving gel. 7. Insert the comb immediately. 8. Allow the gel to polymerize for at least one hour. 9. Prepare samples while the gel is polymerizing. 10. Carefully remove the comb. NOTE: Gels may be stored overnight at 4 C with the comb in place and wrapped in plastic wrap. Casting gradient polyacrylamide gels Hand casting gradient gels is not covered in this resource. Due to the complexity involved in hand casting gradient gels, precast gels have become a popular alternative. We recommend PAGEr Precast Gels (see page 48), or ProSieve Gel Solution (see page 60), which provides gradient quality separation from a linear gel format. Detailed discussions and protocols for preparing gradient gels can be found in Electrophoresis in Practice, 2nd Edition. 158

167 Section X: Protein Separation in Polyacrylamide Gels Loading and Running Proteins on Polyacrylamide Gels Introduction Protein load levels will vary depending upon sample purity and staining method used. For highly purified proteins, 0.5 μg to 5 μg protein per lane on a minigel is generally sufficient. Complete mixtures such as cell lysates may require as much as 50 μg protein per lane. The table below provides lower detection limits for protein detection. Protein stain detection limits Protein Stain Coomassie Blue Stain Silver Stain SYPRO Orange Protein Gel Stain SYPRO Red Protein Gel Stain SYPRO Tangerine Protein Gel Stain Lower Detection Limit (protein/band) 100 ng 1 ng 1 ng2 ng 1 ng2 ng 4 ng8 ng NOTE: Limits are based on optimal detection methods for each stain. The photographs below demonstrate the detection sensitivities of commonly used stains Coomassie Blue Stain (CCD camera) SYPRO Red Protein Gel Stain diluted in 7.5% acetic acid (Polaroid Photo UV light) SYPRO Tangerine Protein Gel Stain diluted in PBS (Polaroid Photo UV light) Silver Stain (Amersham Plus One Kit) Protein Separation in Polyacrylamide Gels SYPRO Ruby Stain Serial dilutions of ProSieve Protein Markers were separated on 12% PAGEr Gold Precast Gels, stained and photographed as noted. The figure shows the staining sensitivity seen for the 50 kda band of the marker. The level of protein present is indicated in nanograms. Staining was performed following the manufacturer s instructions provided with the stains. Exposure times for photography were adjusted to obtain highest possible detection levels. Note that in both silver stain examples shown, development was allowed to proceed for an extended time period relative to the range given in the instructions to maximize detection. In the case of the Amersham kit; this results in overexposure of higher protein loading levels. Images of the silver stained gels were captured using a CCD camera system. 159

168 Section X: Protein Separation in Polyacrylamide Gels Loading Buffers Protein Separation in Polyacrylamide Gels Introduction In general, loading buffers for protein electrophoresis contain Tris-HCl, ph 6.8; 2% SDS; a reducing agent such as dithiothreitol (DTT), -mercaptoethanol ( ME), or Tris[2- Carboxyethylphosphine]hydrochloride (TCEP); glycerol (a sinking agent) and a marker dye. An alternative reducing agent to ME, Bond-Breaker TCEP Solution from Thermo Scientific is an odor-free, ready-to-use solution added to the sample buffer prior to denaturation. Gel loading buffers serve four purposes in protein electrophoresis: Reduction of protein complexes if performing denaturing PAGE Dissociation of proteins to allow them to run through the gel Increase the density of the sample ensuring samples drop evenly into the well Addition of a dye to the sample to simplify loading and monitor the electrophoretic process 2X Tris-Glycine SDS sample buffer 2X concentrate amount to add for 2X concentrate 126 mm Tris-HCl, ph ml of 0.5 M Tris-HCl, ph % Glycerol 2 ml Glycerol 4% SDS 4 ml of 10% SDS 0.005% Bromophenol blue 0.5 ml of 0.1% Bromophenol blue Adjust volume to 10 ml with distilled water (1X = 63 mm Tris-HCl, 10% glycerol, 2% SDS, % Bromophenol blue, 2.5% ME) Sample preparation tips for sample preparation Keep samples on ice prior to adding the sample buffer Add room temperature sample buffer to the cold samples If preparing samples for future use, aliquot treated samples into usable aliquots to avoid freeze thawing Do not leave the samples in SDS sample buffer at room temperature without first heating to 95 C to inactivate proteases. A loss of high-molecular weight bands and general smearing of the bands are indications of protease activity Procedure for sample preparation Follow the guidelines below for preparing protein samples for electrophoresis. 1. Add 0.5 ml ME or 1 ml TCEP to 10 ml of 2X Tris-Glycine SDS sample buffer. 2. Add 1 part 2X sample buffer to 1 part sample on ice. 3. Mix well. 4. Heat sample at 95 C C in a boiling water bath for 4 minutes. 5. Place on ice until ready-to-use or store at 20 C for up to 6 months. Loading the samples tips for loading samples Load the same sample volume in each well If the well is not needed for a sample, load with 1X sample buffer NOTE: If a well is left empty, adjacent samples may spread Materials Boiling water bath Ice Reagents ME or TCEP 10 ml 2X Tris-Glycine SDS sample buffer 160

169 Section X: Protein Separation in Polyacrylamide Gels Loading Buffers continued Procedure for sample loading 1. Slowly and gently lift the comb straight up from the gel. Allow air to enter the well area to release the vacuum which forms between the gel and the comb. 2. Rinse each well with 1X electrophoresis buffer. 3. Place the gel into the electrophoresis chamber. 4. Add 1X electrophoresis buffer to cover the wells. 5. Gently load the desired volume of sample beneath the buffer in each well. NOTE: Loading the sample too fast will lead to diffusion of the sample in the well. Materials Electrophoresis chamber Gel Reagents Samples 1X Electrophoresis buffer Protein Separation in Polyacrylamide Gels Section X: Protein Separation in Polyacrylamide Gels Optimal Voltage, Running Times and Power Settings Optimal voltage Tris-Glycine polyacrylamide minigels are typically run at constant voltage between volts. During electrophoresis, the current drops and heat decreases. Voltage set too high, or not limited causes excessive heating, resulting in band distortion and potential damage to the gel and apparatus. Constant voltage allows the same voltage to be used with multiple gels in an apparatus. Gel thickness is not a factor when using constant voltage. For large format gels, a constant current setting with a voltage limit set slightly higher (5 volts) than the expected voltage for the run may also be used to maintain sample velocity. Optimal electrophoretic time The gel should be run until the bromophenol blue dye has migrated to the bottom of the gel. Gel running times are dependent upon the buffer system used, the length of the gel and the polyacrylamide concentration. Typically minigels will take approximately minutes to run. Whereas large format gels may take as long as 5 hours to run. Section X: Protein Separation in Polyacrylamide Gels ProSieve 50 Gel Solution ProSieve 50 Gel Solution is a modified acrylamide formulation for electrophoresis of large proteins. See page 60 for ordering information. Advantages Gradient separation From easy-to-cast single concentration gels Easy-to-handle Gels are more durable than standard acrylamide Sharp resolution Resolves large proteins (>200 kda) Fast Shorter destaining times and faster protein mobility times Low Background Even when used with Silver Stain 161

170 Section X: Protein Separation in Polyacrylamide Gels PAGEr Gold Precast Polyacrylamide Minigels Protein Separation in Polyacrylamide Gels Introduction PAGEr Gold Precast Gels are Tris-Glycine (Tris-HCl) gels with a 4% stacking gel. The gels contain no SDS, so can be used for native gel electrophoresis or SDS may be added to the sample and running buffer for denaturing conditions. Advantages Eliminates gel preparation time Printed well markings for ease of sample loading Easy open cassette Compatible with most commonly used vertical minigel apparatuses The photograph below demonstrates the resolution performance of PAGEr Gold Precast Gels Separation ranges for proteins in PAGEr Gold Tris-Glycine Gels Polyacrylamide Size Separation Concentration Range 7.5% 50 kda kda 10% 25 kda kda 12% 20 kda kda 15% 10 kda - 50 kda 4-20% 5 kda kda 10-20% 5 kda kda 4-12% 25 kda kda 8-16% 15 kda kda See page 48 for ordering information Separations were run using a 10 well 4-20% Tris-Glycine PAGEr Gold Precast Gel. The gel was run at 120 volts until the tracking dye reached the base of the gel (approximately 90 minutes). ProSieve Protein Markers were loaded in lanes 1, 5 and 10; aqueous extracts of three different strains of E. coli cells were run in lanes 2-4 and 7-9. Lanes 2-4 were loaded at one half the load level of lanes 7-9. Lane 6 contains a mixture of purified proteins. Proteins were detected by Coomassie Blue Stain. 162

171 Section X: Protein Separation in Polyacrylamide Gels PAGEr Gold Precast Polyacrylamide Minigels continued The figure below demonstrates the separation patterns of ProSieve Protein Markers on PAGEr Gold Precast Gels at various concentrations. Smaller molecular weight bands do not separate on some lower concentration gels. Percentage PAGEr Precast Gel Gel Concentration Separation Pattern % 12% 10% 7.5% 10-20% 4-20% 8-16% 4-12% Protein Separation in Polyacrylamide Gels Separation Size Range kda kda kda kda kda kda kda kda ProSieve Protein Markers contain 10 proteins with exact masses of 225, 150, 100, 75, 50, 35, 25, 15, 10 and 5 kda. Gels were run at 175 volts until the dye front reached the bottom of the gel (approximately 60 minutes). 8 ml - 10 ml of ProSieve Protein Marker was loaded per lane (0.8 μg - 1 μg per band). Gels were stained with Coomassie Blue Stain. Chamber Compatibility PAGEr Gels are available in 9 cm 10 cm and 10 cm 10 cm sizes and fit most standard mini-vertical systems. Contact Scientific Support for information about your specific chamber. Standard Vertical Systems PAGEr Gels PAGEr Minigel Chamber 9 cm 10 cm 10 cm 10 cm gels Bio-Rad MiniPROTEAN II, MiniPROTEAN 3 or 9 cm 10 cm gels Ready Gel Cell Systems Reverse the inner core gasket so the at side faces outward. Novex XCell SureLock Mini-Cell 9 cm 10 cm 10 cm 10 cm gels Request the spacer for the XCell SureLock Mini-Cell Chamber from Scientific Support, (Cat. No ). FisherBiotech Vertical Minigel FBVE121, 10 cm 10 cm gels Owl Separations Systems Wolverine P82 Chamber comes with 2 sets of wedges. Use the thinner wedges for the PAGEr Gold Gels. FisherBiotech Vertical Minigel FB-VE101, 10 cm 10 cm gels Owl Separations Systems Penguin Model P8DS Request adaptor for these chambers from Scientific Support, (Cat. No ). Hoefer Mighty Small (SE250) 9 cm 10 cm 10 cm 10 cm gels Replace the buffer chamber with a Deep lower buffer chamber for the SE260, order number , from GE Healthcare. Daiichi 2, ISS chambers 10 cm 10 cm gels See page 164 for modification instructions. Some chambers may require adjustment for optimal fit, see page 164. Standard Vertical Systems Novex XCell II Hoefer Mighty Small (SE260) EC 120 Mini Vertical Gel System Biometra Mini V Chamber CBS Scientific MGV System (10 cm 8 cm units) Sigma-Aldrich Mini Techware (11.3 cm 10 cm units) Zaxis System 2000 Hoefer Mini VE PAGEr Gels 9 cm 10 cm or 10 cm 10 cm gels 9 cm 10 cm or 10 cm 10 cm gels 9 cm 10 cm or 10 cm 10 cm gels 9 cm 10 cm gels 9 cm 10 cm gels 10 cm 10 cm gels 10 cm 10 cm gels 10 cm 10 cm gels 163

172 Section X: Protein Separation in Polyacrylamide Gels Chamber Modification Instructions Protein Separation in Polyacrylamide Gels The following guidelines will ensure optimal performance of PAGEr Gold Precast Gels in these systems. Bio-Rad Mini-PROTEAN II, Mini-PROTEAN III or Ready Gel Cell Systems Remove the rubber gasket from the inner core. Replace the gasket in the reverse orientation into the unit so the at side faces outward. Daiichi 2 To run one gel: Place one cassette on wedge side of chamber. Use the taller half of an Owl glass cassette or an equivalent as a buffer dam on the other side. Use Daiichi wedges. The PAGEr Gold Cassettes cannot be used as the dam in this system. To run two gels: Widen the hole on the yellow port of the inner core. Replace the long arm wedges with modified wedges, which are thicker and shorter. This chamber modification and the new wedges are available from Lonza free of charge. Contact Lonza Scientific Support for details. FisherBioTech Vertical Minigel Protein System: FB-VE10-1 Mini Chamber Lonza offers an adapator for this chamber, contact Lonza Scientific Support for details. The adapter only works if the inner gasket is white. Replace black-plastic side spacer with Lonza adapator. Use one on each side of the inner core. FisherBioTech Vertical Minigel Protein System: FB-VE12-1 Chamber comes with 2 sets of wedges. Use the thinner wedges with PAGEr Gold Precast Gels. Hoefer Mighty Small (SE250) Replace the lower buffer chamber with a Deep Lower Buffer Chamber for the SE260. Available from GE Healthcare (Part No ). The extra depth of the SE260 buffer chamber allows the lid to lock into place. Novex XCell SureLock Mini-Cell Lonza offers a spacer for this chamber, contact Lonza Scientific Support for details. To run one gel: Put the gel in the front of the chamber. Put the buffer dam on the back. Place the Lonza spacer between the buffer dam and the buffer core, on the side of the chamber with the gel tension wedge. Lock the gel tension wedge. To run two gels: Put a gel on each side of the buffer core. Place the Lonza spacer between the gel and the buffer core, on the side of the chamber with the gel tension wedge. Lock the gel tension wedge. Owl Separation Systems Penguin Model P8DS-1 Lonza offers an adaptor for this chamber, contact Lonza Scientific Support for details. The Lonza adaptor for the Penguin model only works if the inner gasket is white. Replace black-plastic side spacer with Lonza adaptor. Use one on each side of the inner core. 164

173 Section X: Protein Separation in Polyacrylamide Gels Chamber Modification Instructions continued Procedure for electrophoresis using PAGEr Gold Precast Gels 1. Cut open pouch and remove gel. 2. Rinse the gel with distilled or deionized water. 3. Slowly and gently lift the comb straight up. NOTE: Put the comb aside so it can be used to separate the cassette plates at the end of the run. 4. Remove white tape from bottom of the cassette. NOTE: The sharp end of the comb can be used to peel off the tape. 5. Mount the cassette(s) into the electrophoresis chamber so the printed side faces the outer (anode) buffer chamber. If running only one gel, mount the appropriate buffer dam. 6. Fill the buffer chambers with 1X running buffer. 7. Wash the wells with 1X running buffer, displacing any air bubbles in the wells. 8. Load samples. NOTE: For best results, load 1X sample buffer in the wells without samples. 9. Run the gels at a constant voltage of volts until the dye front is near the bottom of the gel (approximately minutes). 10. Remove the gel(s) from electrophoresis chamber. 11. Place the cassette on a flat surface with the short side of the cassette facing up. 12. Using the end of the comb and starting at the top of the cassette separate the two plates by using a twisting motion to crack the cassette. 13. Carefully remove the short plate. 14. Hold the plate with the gel over an open container. If the gel is adhered to Large Plate Small Plate Then Run end of comb or a gloved finger behind slot on cassette to push out the lip at the bottom of the gel. Use the comb or a spatula to loosen one lower corner of the gel. 15. Allow gel to peel away and gently drop into the container. 16. Fix, stain and destain or blot the gel as desired. Materials Scissors Pipette Electrophoresis apparatus Power Supply Container Spatula Reagents Distilled water Running Buffer AccuGENE 10X Tris-Glycine or Tris- Glycine SDS Buffer PAGEr Gold Precast Gels can be run at higher voltages to achieve faster run times. If using Tris-glycine Running Buffer gels can be run at volts for approximately 40 minutes. If using Tris-Tricine running buffer gels can be run at volts for approximately 30 minutes. The photographs below show the separation of protein samples on PAGEr Gold Precast Gels under increased voltage. kda % PAGEr Gold Gel Tris-Glycine kda % PAGEr Gold Gel Tris-Tricine Samples were prepared using Tris-Glycine SDS Sample Buffer and -Mercaptoethanol as a reducing agent. Lane 1: ProSieve Protein Markers; Lanes 2&3: E. coli extracts; Lane 4: Bovine Serum Albumin (BSA). Gel 1: 4-20% gel in 1X Tris-Glycine SDS Buffer run at 230 volts for 40 minutes. Gel 2: 8-16% gel in 1X Tris-Tricine SDS Buffer run at 185 volts for 30 minutes. Samples were run until the tracking dye reached the base of the gels. Proteins were detected using Coomassie Blue Stain. Protein Separation in Polyacrylamide Gels 165

174 Section X: Protein Separation in Polyacrylamide Gels Detection of Proteins in Polyacrylamide Gels Protein Separation in Polyacrylamide Gels Detecting proteins with SYPRO Protein Gel Stains SYPRO Protein Gel Stains are highly sensitive ffuorescent stains for the rapid detection of proteins in polyacrylamide gels. These stains can detect as little as 4 ng - 8 ng of protein per band in minutes without destaining. Gels stained with SYPRO Protein Gel Stains exhibit low background and minimal protein-to-protein staining variability. Gels can be documented using standard imaging systems. Tips for staining gels with SYPRO Protein Gel Stains The SDS front at the bottom of the gel stains heavily with SYPRO Stains. Unless the protein of interest comigrates with the SDS front, we recommend running the SDS front off the end of the gel Colored stains such as Coomassie Blue Stain and colored protein markers may interfere with SYPRO staining and quench ffuorescence. To stain gels previously stained with Coomassie Blue Stain, soak the gel in several changes of 7.5% acetic acid to remove the Coomassie Stain. Then incubate the gel in 0.05% SDS for 30 minutes and stain with SYPRO Stain as usual Glove powder can leave background markings on gels. Rinse or wash gloves prior to handling gels Clean the surface of the transilluminator after each use with deionized water and a soft cloth. Fluorescent stains (such as SYPRO Stains or ethidium bromide) can accumulate on the transilluminator and may cause high background Handle gels carefully to avoid non-specific staining of areas of the gel that have been squeezed SYPRO Stains may be photobleached after several minutes of exposure to UV light. If a gel becomes photobleached, restain by incubating in the staining solution SYPRO stained gels can be restained with Coomassie Blue or Silver Stain procedures Plastic wraps and GelBond Film will autoffuoresce when exposed to UV light resulting in very high background. Gels backed with GelBond Film can be photographed by inverting the gel on the transilluminator Select the Best Stain for your Application Application SYPRO Ruby SYPRO Tangerine SYPRO Red High performance staining Staining prior to Western blotting 2D electrophoresis Edman microsequencing Mass spectrometry Quantitation Zymography Electroelution Membrane staining Protein expression Detection prior to Immunostaining Difficult to stain proteins IEF Gels Materials Clear polypropylene container (e.g., Rubbermaid Recycling #5 Plastics) Reagents 7.5% (v/v) acetic acid SYPRO Protein Gel Stain stock solution Caution: Materials and methods shown here present hazards to the user and the environment. Refer to the safety information on page 210 before beginning these procedures. Caution: Acetic acid causes burns and respiratory irritation. Take precautions to prevent exposure. 166

175 Section X: Protein Separation in Polyacrylamide Gels Detection of Proteins in Polyacrylamide Gels continued General Procedure for staining proteins with SYPRO Protein Gel Stains 1. Run SDS-polyacrylamide gels according to standard protocols. NOTE: To reduce background staining with SYPRO Red and Tangerine Stains, use 0.05% SDS in the running buffer. Gels run in 0.05% SDS show no change in the migration pattern of proteins. 2. Dilute the 5,000X concentrate to a 1X solution, in 7.5% (v/v) acetic acid, in a clear plastic polypropylene container. For example, for every 10 ml acetic acid add 2 μl stock SYPRO Stain solution. 3. Mix well. 4. Place the gel into the staining container and cover with a lid to protect from light. 5. Gently agitate the gel at room temperature. 6. Stain the gel for minutes. NOTE: The optimal staining time depends on the thickness of the gel and the gel concentration. Longer staining times may be required as gel thickness increases. 7. Briefly rinse the gel in 7.5% acetic acid. 8. Remove the gel from the staining container and photograph the gel following the procedure on page 168. Procedure for staining nondenaturing gels with SYPRO Red Protein Gel Stain Options for staining proteins with SYPRO Red Stain after native gel electrophoresis: 1. Dissolve the stain 1:5,000 in distilled water and follow the general protocol for staining proteins with SYPRO Protein Gel Stains. This will be highly protein-selective, and will be less sensitive than staining proteins in SDS gels. To increase the signal, a long film exposure can be used since the background fluorescence is essentially zero. 2. Soak the gel after electrophoresis in 0.05% SDS for 30 minutes, then stain with a 1:5,000 solution of SYPRO Stain diluted in 7.5% acetic acid. Proteins will be denatured and fixed after electrophoresis using this treatment. Procedure for staining proteins with SYPRO Tangerine Protein Gel Stain: Non-fixing protocol SYPRO Tangerine Protein Gel Stain is an extremely versatile uorescent stain that does not alter protein structure, interfere with the transfer of proteins to blotting membranes, or use organic solvents. The stain can be diluted into a wide range of buffers with a ph range from 4-10 and if fixing is necessary, it can be diluted into 7.5% acetic acid following the General Protocol for Staining Proteins with SYPRO Gel Stains. If proteins are to be used for subsequent analysis, dilute SYPRO Tangerine stock solution into 50 mm phosphate, 150 mm NaCl, ph 7.0 or use one of the buffers listed below prepared as 50 mm mm solutions containing 150 mm NaCl. Compatible Buffers Formate ph 4.0 HEPES ph 7.5 Citrate ph 4.5 Tris acetate ph 8.0 Acetate ph 5.0 Tris-HCl ph 8.5 MES ph 6.0 Tris borate, 20 mm EDTA ph 9.0 Imidazole ph 7.0 Bicarbonate, ph 10.0 Materials Clear polypropylene container (e.g., Rubbermaid Recycling #5 Plastics) Reagents 50 mm Phosphate, 150 mm NaCl, ph 7.0 or suitable buffer Caution: Materials and methods shown here present hazards to the user and the environment. Refer to the safety information on page 210 before beginning these procedures. Protein Separation in Polyacrylamide Gels 167

176 Section X: Protein Separation in Polyacrylamide Gels Detection of Proteins in Polyacrylamide Gels continued Protein Separation in Polyacrylamide Gels Follow this procedure if the gel will be used for subsequent analysis such as zymography or Western blotting. Stained proteins can also be eluted from gels and used for further analysis such as mass spectrometry. 1. Run SDS-polyacrylamide gels according to standard protocols. It is not necessary to decrease the amount of SDS present in the running buffer when using SYPRO Tangerine Gel Stain. 2. Dilute the 5,000X concentrate to a 1X solution, in 7.5% (v/v) acetic acid solution, in a clear plastic polypropylene container. For example, for every 10 ml acetic acid add 2 μl stock SYPRO Stain solution. 3. Mix well. 4. Place the gel into the staining container and cover to protect from light. 5. Gently agitate the gel at room temperature. 6. Stain the gel for minutes. The optimal staining time depends on the thickness of the gel and the gel concentration. Longer staining times may be required as gel thickness increases. 7. Remove the gel from the staining container and immediately photograph the gel following the procedure on page 168. NOTE: If the gel will be blotted after staining with SYPRO Tangerine Stain, stain the gel according to the procedure above, using 50 mm phosphate, 150 mm NaCl as the stain diluant. Add 0.1% SDS to the transfer buffer. This will help in the transfer of some proteins to the membrane. Viewing gels stained with SYPRO Protein Gel Stains All SYPRO Protein Gel Stains have two excitation wavelengths; in the UV region at approximately 300 nm and in the blue range of visible spectrum, between 470 and 610 nm. The stains can be visualized using a UV transilluminator or the Dark Reader Transilluminator. Photographing gels stained with SYPRO Protein Gel Stains Protein bands stained with SYPRO Protein Gel Stains are best seen by photo graphing the gel. The integrating effect of a camera or imaging system can detect bands that are not visible to the eye. Gels stained with SYPRO Protein Gel Stains can be photographed using Polaroid Cameras, CCD camera systems or laser scanners. The highest sensitivity using a Polaroid Camera can be obtained using Polaroid 667 black-and-white print film and the SYPRO Protein Gel Stain Photographic Filter. Exposure time will vary with the intensity of the illumination source. Begin with an f-stop of 4.5 and an exposure of 2-8 seconds. Use of an ethidium bromide filter is not recommended as it blocks much of the light and leads to lower detection sensitivity The SYPRO Photographic Filter does not work with CCD Camera systems. For CCD cameras, use the emission and excitation data below and check with the camera manufacturer for the appropriate filter Stain Emission (nm) Excitation (nm) SYPRO Red and 550 SYPRO Orange and 470 SYPRO Tangerine and 490 Detecting proteins with Coomassie Blue Stain Coomassie Blue Stain binds nonspecifically to most proteins. Proteins are fixed and stained in a Coomassie Blue staining solution and subsequently destained to eliminate the blue background from the gel. Gels can be dried, photographed or stored wet. Coomassie Blue Stain solution 1X Working Solution Amount for 1X working Solution* 40% Ethanol 400 ml Ethanol 0.125% Coomassie Blue 1.25 g Coomassie Blue R-250 Distilled water 500 ml Distilled water 10% Acetic acid 100 ml Acetic acid *Add reagents in the order provided Coomassie Blue destain solution 1X Working Solution Amount for 1X working Solution* 5% Ethanol 50 ml Ethanol 7.5% Acetic acid 75 ml Acetic acid Adjust volume to 1 liter with distilled water Caution: These solutions should be prepared under the fume hood. 168

177 Section X: Protein Separation in Polyacrylamide Gels Detection of Proteins in Polyacrylamide Gels continued Procedure 1. Prepare enough Coomassie Blue Staining Solution to cover the surface of the polyacrylamide gel. 2. Prewarm the Coomassie Blue Stain Solution to 55 C. 3. Remove the gel from the glass plates. 4. Submerge the gel in the Coomassie Blue Stain Solution. 5. Gently agitate the gel for 20 minutes at 55 C until the gel becomes blue. Alternatively, solutions may be heated to 45 C and incubated at room temperature. 6. Decant stain solution from container. NOTE: Coomassie Blue Staining Solutions can be saved and stored to stain multiple gels. As the stain solution reaches its use limit, gels will appear grainy and will not destain completely and new staining solutions should be prepared. 7. Briefly rinse excess stain from gel in water 8. Transfer the gel into the destain solution. 9. Gently agitate the gel at 55 C until the gel has destained and bands are visible. (Approximately 1 hour). NOTE: The destain solution may need to be changed occasionally until the background is clear. Do not over destain, which can lead to loss of band intensity. Pieces of paper towel or a Kimwipe can be placed in the corner of the container to speed up this process. Change the tissues when they are saturated with Coomassie Blue Stain. Materials Kimwipes or paper towels 3 plastic containers with covers Shaking incubator set to 55 C Reagents Stain solution 0.125% Coomassie Blue, 40% Ethanol, 10% Acetic acid Destain solution 5% Ethanol, 7.5% Acetic acid Detecting proteins with silver stain Silver staining is based on differential reduction of silver ions bound to sulfhydryl and carboxyl side chains on proteins. After electrophoresis, proteins are fixed, exposed to silver nitrite and developed to form a black precipitate of silver. All silver staining procedures are time consuming. Many good kits are commercially available which make the procedure faster and easier. The BioRad Silver Stain Plus Kit is recommended for its versatility and ease of use. The procedure described below is a modification of Morrisey. This procedure uses dithiothreitol (DTT) to improve reproducibility. An advantage of this version is that development occurs more slowly than many silver staining protocols, giving more control over the final image. Procedure Wear gloves and use only glass-distilled water and glass staining containers. All steps can be done at room temperature. 1. Gently agitate the gel in 100 ml Destain I for 30 minutes to overnight. 2. Remove Destain solution I. 3. Gently agitate the gel in 100 ml of Destain II for 30 minutes. 4. Remove Destain II. 5. Gently agitate the gel in 100 ml cross-linking solution for 30 minutes. NOTE: For small peptides, incubate with glutaraldehyde overnight to insure retention of the peptides in the gel. 6. Remove cross-linking solution. 7. Wash gel with several changes of water over a 2 hour period. NOTE: Alternatively, the gel can be placed in 2 liters of water and the next morning washed for 30 minutes in fresh water. High background will result if the glutaraldehyde is not completely washed out of the gel. 8. Gently agitate the gel in DTT solution for 30 minutes. 9. Remove DTT solution and drain well, but do not rinse the gel. 10. Gently agitate the gel in 100 ml silver nitrate solution for 30 minutes. Protein Separation in Polyacrylamide Gels Continued on next page. 169

178 Section X: Protein Separation in Polyacrylamide Gels Detection of Proteins in Polyacrylamide Gels continued Protein Separation in Polyacrylamide Gels 11. Place the staining tray under running deionized water and swirl for a few seconds. 12. Remove the water. 13. Add 50 ml of Developing solution, swirl briefly, then discard the solution. 14. Repeat step 13. NOTE: This reacts with excess silver and prevents non-specific staining of the gel. 15. Add 100 ml of Developing solution and shake slowly. NOTE: Staining occurs slowly at first but then progresses rapidly. Development takes approximately 5 to 10 minutes. 16. When the bands are slightly lighter than the desired staining level, remove developing solution, rinse quickly with water, add Destain II to cover the gel, as the stop solution. Alternatively, add 5 ml of Stop Solution to the developer to stop development. In either case, development will not stop immediately but continues for approximately 5 minutes after adding Stop Solution. 17. Wash the gel several times in Destain II. 18. Rinse the gel with water. 19. Store in water or dry the gel. Materials Glass containers Orbital shaker References Ausubel, F.M., et al., Current Protocols in Molecular Biology, Wiley and Sons, Hoeffer Scientific Instruments, Protein Electrophoresis Applications Guide, Lauber, W.M., et al., Electrophoresis, 22: , Malone, J. P., et al., Electrophoresis, 22: , Morrissey, J.H., Anal. Biochem., 117: , Sambrook, J., et al., Molecular Cloning; A Laboratory Manual, 2nd edition, Cold Spring Harbor Laboratory Press, Davis, B.J., Ann. N.Y. Acad. Sci., 121: , Dunn, M.J., Gel Electrophoresis: Proteins, Bios Scientific Publishers Limited, Hames, B.D., et al., Gel Electrophoresis of Proteins. A Practical Approach, Oxford University Press, Harlow, E. and Lane, D., Antibodies: A Laboratory Manual, Cold Spring Harbor Laboratory, Laemmli, U.K., Nature, 227: , Ornstein, L., Ann. N.Y. Acad. Sci., 121: , Schaegger, H. and von Jagow, G.V., Anal. Biochem., 166: , Westermeier, R., Electrophoresis in Practice: A Guide to Methods and Applications of DNA and Protein Separations, 2nd edition, VCH, A Wiley Company, Wyckoff, M., et al., Anal. Biochem., 78: , Reagents Glass distilled water Destain I (40% Methanol 7% Acetic acid) 400 ml Methanol 70 ml Acetic acid Adjust volume to 1 liter with distilled water Store at room temperature indefinitely Destain II (5% Methanol, 7% Acetic acid) 700 ml Acetic acid 500 ml Methanol Adjust volume to 10 liters with distilled water Store at room temperature indefinitely Cross-linking solution (10% glutaraldehyde) 20 ml of 50% glutaraldehyde stock in 100 ml of distilled water Dithiothreitol (DTT) Solution (5 mg/ml) 5.0 mg DTT in 1 liter of distilled water Silver Nitrate Solution (0.1% w/v Silver nitrate) Caution: Glutaraldehyde is toxic and must be handled in a fume hood 1.0 g Silver nitrate (AgNO3) in 1 liter of distilled water 3% Sodium Carbonate (3% w/v) 60.0 g Sodium carbonate in 2 liters of distilled water Store in glass container Developing Solution (3% Sodium carbonate, 0.019% Formaldehyde) 200 ml of 3% Sodium carbonate 100 ml of 37% Formaldehyde Prepare just prior to use Stop Solution (2.3 M Sodium citrate) g Sodium citrate, dihydrate (FW 294.1) Adjust volume to 100 ml in distilled water Caution: Materials and methods shown here present hazards to the user and the environment. Refer to the safety information on page 210 before beginning these procedures. 170

179 Section XI: Blotting Proteins from Polyacrylamide Gels Blotting Proteins from Polycrylamide Gels In This Section Introduction 172 Semi-dry Blotting PAGEr Gold Precast Gels 173 Tank Blotting PAGEr Gold Precast Gels 174 References

180 Section XI: Blotting Proteins from Polyacrylamide Gels Introduction Protein transfer efficiency in blotting applications is dependent upon multiple factors, including gel percentage, gel thickness, protein size, transfer conditions (e.g., buffer and voltage), and type and quality of membrane. To achieve optimal transfer efficiency, transfer conditions must be adjusted to address these varying factors. Blotting Proteins from Polycrylamide Gels Choosing the appropriate membrane Nitrocellulose PVDF Nylon Hydrophobic binding Hydrophobic binding Hydrophobic & electrostatic binding General purpose membrane SDS tolerant Stable if baked Low background High background High background Low strength High strength High strength Becomes brittle if baked Suitable for protein sequencing Least suitable for Western transfer Transfer solutions Formula for Towbin transfer solution: Towbin Transfer Solution 1X Working Solution Amount for 1X Working Solution 25 mm Tris base 30.3 g Tris base 192 mm Glycine g Glycine 0.1% SDS 10.0 g Adjust volume to 8 liters with distilled water Measure, but do not adjust ph; it should be approximately 8.2 to % Methanol 2 L Methanol Adjust volume to 10 liters with distilled water It may be necessary to lower the concentrations of methanol, SDS or both to obtain the optimal balance of transfer and binding efficiency. The table below outlines the effects that SDS and methanol have on protein transfer. SDS Improves transfer of proteins >60 kda Decreases binding efficiency Not compatible with nylon membranes Include 0.1% - 0.2% in transfer buffer Methanol Improves binding efficiency Decreases transfer efficiency Do not soak gel in transfer buffer prior to blotting Include 20% in transfer buffer 172

181 Section XI: Blotting Proteins from Polyacrylamide Gels Tips for increasing transfer efficiency Use the lowest concentration gel that will resolve the protein(s) of interest. Avoid using gels 1 mm thick. Thick gels may require longer blotting times. Decrease the concentration of methanol to optimize transfer efficiency of proteins >150 kda. Small proteins tend to transfer more easily than large proteins. Longer transfer times may be used to ensure complete transfer of large proteins (>60 kda), proteins from native gels, and thicker gels. Use two membranes if transferring for an extended period of time (>1 hour). The second membrane will bind any protein that may transfer through the first. This can be verified by membrane staining. Use ProSieve Color Protein Markers (see page 51) to confirm that transfer has occurred to the membrane and not the filter paper. Use a chopping motion when removing the well and foot area. Slicing the gel may cause tearing. Gently roll out air bubbles between transfer stack layers using a wet glass rod or pipette. If the gel sticks to the filter paper or membrane after transfer, soak for 5-15 minutes in water then gently peel the filter paper away. If proteins are left in the gel after following recommended transfer conditions, increasing the voltage by no more than 5 volts may be helpful. Blotting Proteins from Polycrylamide Gels Section XI: Blotting Proteins from Polyacrylamide Gels Semi-dry Blotting PAGEr Gold Precast Gels This protocol is for use with the BioRad Trans-Blot Semi-Dry Cell. If using another manufacturer s blotting apparatus, follow manufacturer s instructions for use. Tips for Semi-Dry Blotting To prevent the stack from drying out, add extra transfer solution (2 ml - 5 ml) to the top layer of filter paper before closing the lid on the blotting apparatus. Center the gel on the membrane. Occasionally, the gel will overlap the membrane and stick to the filter paper below. If this occurs, gently break the seal with a scalpel. Procedure for semi-dry blotting NOTE: Nitrocellulose or PVDF membranes are recommended for this method and should be wetted in methanol prior to wetting in transfer solution. 1. Electrophorese gel following standard procedure. 2. Carefully trim off stacking gel and bottom foot from the gel. 3. Soak gel for 20 minutes in chilled 1X transfer solution. 4. Cut the filter paper, blot paper and membrane to the size of the gel. NOTE: Transfer membranes should be handled at the edges with gloves worn. 5. Soak membrane, blotting paper and filter paper for 5-10 minutes in 1X transfer solution. 6. To make a semi-dry blotting sandwich, stack in the following order: 6a. Mask 6b. Prewet extra thick blot paper 6c. Prewet nitrocellulose membrane 6d. Polyacrylamide gel. 6e. Prewet Whatman Grade 114 or 54 Filter Paper 6f. Prewet extra thick blot paper 6g. Top platen (stainless steel cathode), and safety cover 7. Turn on the power and transfer at 25 volts (constant) at 400 ma for: 60 minutes for 10 kda kda proteins 90 minutes for 100 kda kda proteins NOTE: Optimized conditions will be required for different proteins or different membranes. Continued on next page. 173

182 Section XI: Blotting Proteins from Polyacrylamide Gels Semi-dry Blotting PAGEr Gold Precast Gels continued Materials and Reagents Transfer membrane Mask Scalpel or razor blade Blotting Proteins from Polyacrylamide Gels A piece of GelBond Film (see page 61), or similar polyester film, the size of the bottom anode, with a rectangular hole the size of the gel cut out of the center. The purpose of the mask is to focus current through the gel stack. Whatman Grade 114 or 54 Filter Paper Extra thick blot paper Scissors Glass rod or pipette 1XTowbin transfer solution at 4 C Shallow tray for soaking membranes and filter paper Section XI: Blotting Proteins from Polyacrylamide Gels Tank Blotting PAGEr Gold Precast Gels For hydrophobic proteins or proteins >100 kda, tank blotting is preferable to semi-dry blotting because prolonged transfers are possible without gel drying. For more detailed information and protocols concerning tank blotting, consult the blotting apparatus manufacturer s instructions. Procedure for tank blotting NOTE: Transfer is performed at 4 C. NOTE: Nitrocellulose or PVDF membranes are recommended for this method and should be wetted in methanol prior to wetting in transfer solution. 1. Electrophorese gel following standard procedures. 2. Carefully trim off stacking gel and bottom foot from the gel. 3. Soak gel for 20 minutes in chilled 1X Transfer solution. 4. Cut the filter paper, blotting paper and membrane to the size of the gel. NOTE: Transfer membranes should be handled at the edges with gloves worn. 5. Soak fiber pads, nitrocellulose membrane and blotting paper for 2 minutes in 1X Transfer solution. 6. To make a tank blotting sandwich, stack in the following order: 6a. Cathode unit 6b. 1 prewet fiber pad 6c. 1 sheet prewet extra thick blot paper 6d. 1 sheet prewet Whatman 114 or 54 Blotting Paper 6e. Polyacrylamide gel 6f. Prewet nitrocellulose membrane 6g. 1 sheet prewet extra thick blot paper 6h. 1 prewet fiber pad 6i. Anode unit 7. Place in tank with nitrocellulose membrane closest to anode (+). 8. Cover with chilled 1X Transfer solution. 9. Turn on the power and transfer by running at 100 volts (constant) at 400 ma for: 90 minutes for 10 kda100 kda proteins 120 minutes for 100 kda300 kda proteins NOTE: Optimized conditions will be required for different proteins or different membranes. Continued on next page 174

183 Section XI: Blotting Proteins from Polyacrylamide Gels Tank Blotting PAGEr Gold Precast Gels - continued Materials Whatman Grade 114 or 54 Filter Paper Extra thick blot paper Transfer membrane Fiber or foam pad Scissors Glass rod or pipette Scalpel or razor blade 1X Towbin transfer solution at 4 C Shallow tray for soaking membranes and filter paper Monitoring protein transfer Protein transfer can be monitored by staining the gel following transfer (see page 166), and/or staining the membrane. Membrane staining should only be performed when duplicate samples have been run on a gel and the membrane can be cut in half, or when a second membrane has been used. Stains commonly used for this purpose include SYPRO Ruby Protein Blot Stain, (see page 55), India ink stain or colloidal gold stain. Described below is the use of the GE Healthcare AuroDye Forte Kit which is a colloidal gold stain. Staining membranes with the GE Healthcare AuroDye Forte Kit This method is compatible with PVDF and nitrocellulose membranes. For more detailed information and protocols, consult the instructions provided with the kit. 1. Place the membrane in a clean glass dish. 2. Add 1X PBS, 0.3% Tween Shake slowly on a shaker for 30 minutes at 37 C. 4. Remove solution and replace with fresh 1X PBS, 0.3% Tween Shake slowly on a shaker for 5 minutes at room temperature. 6. Repeat steps 4 5 two more times. 7. Briefly rinse the membrane in distilled water. 8. Place the membrane in a thermal seal pouch with 15 ml - 20 ml AuroDye Forte Kit for 2-4 hours at room temperature. 9. After fully developed, rinse briefly with distilled water and air dry. Blotting Proteins from Polyacrylamide Gels Materials and Reagents Previously blotted membrane Orbital shaker set to 37 C Orbital or rocking platform shaker Glass dish Thermal seal pouch and sealing unit Distilled water 1X PBS, 0.3% Tween 20 References Ausubel, F.M., et al., Current Protocols in Molecular Biology, Wiley & Sons, Sambrook, J., et al., Molecular Cloning: A Laboratory Manual, 2nd Edition, Cold Spring Harbor Laboratory Press,

184 Notes 176

185 Section XII: Isoelectric Focusing of Proteins on Agarose Gels Isoelectric Focusing of Proteins on Agarose Gels In This Section Introduction 178 Preparation of Agarose Isoelectric Focusing Gels 179 Running Agarose IEF Gels 182 Staining Proteins in Agarose IEF Gels 184 Press Blot Transfer of Proteins from Agarose IEF Gels 188 Preparative Isoelectric Focusing 189 Immunofixation/Immunoperoxidase or Autoradiography 190 Direct Tissue Isoelectric Focusing 191 Resolution Reference Guide 192 References

186 Section XII: Isoelectric Focusing of Proteins on Agarose Gels Isoelectric Focusing of Proteins on Agarose Gels Introduction Separation of proteins in complex mixtures for analytical resolution can be achieved by isoelectric focusing (IEF), in which proteins are separated based on their net charge (isoelectric point, or pi) in the presence of a ph gradient. Analytical focusing is carried out either in polyacrylamide gels most recently prepared with immobilized ph gradients or in agarose gels prepared with mobile carrier ampholytes, which form a ph gradient when subjected to electrophoresis. Separation in agarose gels is more rapid, since the pores of the agarose gel are larger than those of polyacrylamide gels. Advantages No toxic monomer solutions are required Separation of proteins larger than 2,000 kda Shorter staining times Nontacky and compressible (blottable) No catalysts to interfere with separation Applications Immunofixation directly in the gel Crossed immunoelectrofocusing Direct tissue isoelectric focusing Preparative isoelectric focusing Compatible agaroses Isoelectric focusing on agarose gels requires the use of an agarose that has no measurable electroendosmosis (EEO). Lonza has developed two products that can be used for this application, specifically IsoGel Agarose and IsoGel Agarose IEF Plates. IsoGel Agarose is a highly purified agarose that is easy to prepare and produces a less restrictive gel than polyacrylamide, allowing rapid focusing of high molecular weight proteins (>2,000 kda). IsoGel Agarose IEF Plates are ready-to-use 125 mm x 100 mm precast gels that eliminate gel preparation time and provide easy handling throughout IEF processing. The photograph below demonstrates the separation performance of proteins focused on an IsoGel Agarose IEF Plate. Alternatively, Lonza offers IsoGel Agarose IEF Precast Plates. Equ Am 178

187 Section XII: Isoelectric Focusing of Proteins on Agarose Gels Preparation of Agarose Isoelectric Focusing Gels Suggested anolytes and catholytes When selecting anolytes and catholytes for any ph gradient, it is important to closely bracket the ends of the ph range of the ampholytes. Avoid creating ph discontinuities between the ends of the ampholyte range and the bracketing electrolytes. NOTE: ph is dependent on temperature. 25ºC ph values are provided for selection of electrolytes. Under running conditions, the ph will be slightly higher. Anolyte Concentration ph (25ºC) Phosphoric acid 1 M 1.0 Sulfuric acid 0.2 M 1.6 Acetic acid 0.5 M 2.6 L-glutamic acid 40 mm 3.2 Indole acetic acid 3 mm 3.8 L-tyrosine 4 mm 4.5 Catholyte Concentration ph (25ºC) Threonine 50 mm 5.8 Glycine 50 mm 6.15 Hepes 0.4 M 7.3 L-Histidine (free-base)* 40 mm 7.35 Bicine 0.1 M 8.0 Sodium hydroxide 1 M 13.0 *Do not substitute Histidine HCl for free-base. Preparation of the gel casting assembly 1. Spread a few drops of distilled water or 0.1% nonionic detergent on one glass plate. 2. Lay a sheet of GelBond Film, cut slightly smaller than the glass plate, onto the plate with the hydrophilic side up. NOTE: Water droplets spread on the hydrophilic side but bead up on the hydrophobic side of the film. 3. Cover the GelBond Film with a sheet of blotting paper or the interleaving paper supplied with the film. 4. Firmly roll with a rubber roller or wipe with tissues to squeeze out any air bubbles and excess fluid from behind the GelBond Film. 5. Carefully wipe off any excess liquid at the edges. 6. Place the U-frame spacer on top of the GelBond Film. If a U-frame spacer is unavailable, place two spacers on the GelBond Film along either side and one spacer across the bottom edge. 7. Place the second glass plate over the spacer(s). 8. Clamp the assembly with the stationery clamps, using 2 clamps per side and bottom. 9. Warm the cassette in a 55ºC forced hot air oven for 15 minutes. NOTE: GelBond Film may warp if the cassette is heated too long or above 75ºC. Materials GelBond Film (110 mm x 125 mm, Cat. No ) Blotting paper or interleaving paper supplied with Gelbond Film Two thick glass plates (110 mm x 125 mm) A plastic 0.8 mm-thick U-frame spacer or 3 single spacers, 0.8 mm-thick Six stationery binder clamps Blotting paper, rubber roller or tissues Forced hot air oven set to 55ºC Reagents Distilled water or 0.1% nonionic detergent Isoelectric Focusing of Proteins on Agarose Gels 179

188 Section XII: Isoelectric Focusing of Proteins on Agarose Gels Preparation of Agarose Isoelectric Focusing Gels continued Isoelectric Focusing of Proteins on Agarose Gels The following procedure is to prepare a 10 ml IsoGel Agarose gel. Solution preparation 1. Choose a 50 ml beaker or Erlenmeyer flask. 2. Add 8 ml distilled water and a stir bar to the flask or beaker. 3. Premeasure 0.1 g IsoGel Agarose. 4. Premeasure 1.0 g d-sorbitol. 5. Sprinkle in the premeasured agarose powder, while the solution is rapidly stirring. 6. Using a spatula, break up and disperse any agarose clumps and scrape down any powder adhered to the walls of the flask. 7. Add the d-sorbitol while the solution is rapidly stirring. 8. Remove the stir bar. 9. Follow the procedures on page 83 for dissolving agarose. 10. Cool the solution to approximately 60ºC. 11. Add 0.63 ml of ampholyte solution with a 1-cc syringe. 12. Stir the solution well to mix. 13. Maintain the agarose solution at 60 C - 65ºC until casting. 14. Correct for evaporation by adding warm distilled water immediately before gel casting. Materials Erlenmeyer fiask or beaker (50 ml) Microwave Boiling water bath or hot plate Magnetic stir plate Magnetic stir bar 1-cc tuberculin syringe Water bath set to 60 C 20-cc syringe Prewarmed cassette assembly Parafilm or tape Spatula Reagents IsoGel Agarose (see page 56) Distilled water Ampholytes D-sorbitol Boiling distilled water 60 C distilled water 180

189 Section XII: Isoelectric Focusing of Proteins on Agarose Gels Preparation of Agarose Isoelectric Focusing Gels continued Gel casting instructions 1. Flush a 20 cc syringe with boiling water to thoroughly heat it. 2. Expel all water from the barrel and needle. 3. Immediately fill the syringe with the warmed agarose solution. 4. Slowly inject the agarose solution into the pre-warmed cassette. NOTE: Try to avoid introducing air bubbles into the cassette by injecting the solution in a slow-steady stream. 5. Fill the cassette assembly to the top with agarose solution. 6. Seal the top of the cassette with Parafilm or tape to prevent evaporation. 7. Allow the casting assembly to cool at room temperature. 8. Place the gel at 4ºC for one hour. Disassembly of casting cassette 1. Remove the tape and clamps. 2. With the cassette lying flat, insert a flat spatula between the glass plates. 3. Twist gently to break the seal. 4. Carefully remove the top plate, leaving the gel and the GelBond Film attached to the back plate. 5. Remove the spacers. 6. Lift the GelBond Film from the back plate by inserting a flat spatula under the film. 7. Gently lift film and gel away from glass plate. Sample Preparation Successful isoelectric focusing, in part, depends upon the condition of the sample. Situations such as insolubility or high salt content, particularly in the case of high sample loading, should be addressed before the sample is loaded onto the gel. Listed below are general guidelines for sample treatment. High Salt Content: Dialyze the sample against distilled water, 1% glycine, or 0.05 M M ammonium bicarbonate solution. Dissociation of protein aggregates, subunit assemblies or to unfold peptide chains: Add urea to a final concentration of 4 M - 9 M to both the sample and the gel. Samples that are hydrophobic or poorly soluble at or near their pi point: Add either nonionic or zwitterionic detergents to the sample and the gel at a final concentration of 0.05% - 1.0%. Detergents should be added to the agarose solution once the agarose has been dissolved and cooled to 60 C. Nonionic detergents Triton X-100 Zwitterionic detergents CHAPS (available from Sigma Chemical Co., St. Louis, MO) Nonidet (NP-40) Zwittergent 3-14 (available from Calbiochem/ Behring, LaJolla, CA) Tween 80 Isoelectric Focusing of Proteins on Agarose Gels 181

190 Section XII: Isoelectric Focusing of Proteins on Agarose Gels Running Agarose IEF Gels Isoelectric Focusing of Proteins on Agarose Gels Caution: Make certain the power supply is turned off before proceeding. Procedure for gel placement 1. Set the refrigerated circulator bath to 10 C - 15ºC. NOTE: To prevent condensation on the gel and platen, do not circulate the coolant to the IEF chamber just prior to focusing. 2. Spread 0.2 ml ml of distilled water on the cooling platen of the IEF chamber. 3. Lower the gel (film-side down) onto the wetted area. Avoid trapping air under the GelBond Film. 4. Wipe excess fluid from the edges of the film. 5. Blot the surface of the gel briefly with a sheet of finegrained blotting paper. 6. If necessary, trim the edges of the gel parallel to the direction of focusing with a razor blade to ensure the edges are even and free of cracks or small tears. Procedure for electrode wick application 1. Cut two electrode wicks to the exact width of the gel or slightly shorter. 2. Completely immerse one wick in catholyte solution. 3. Place the wick on blotting paper to remove excess fluid. 4. Place on the negative electrode contact of the gel. 5. Completely immerse a second wick in anolyte solution. 6. Place the wick on blotting paper to remove excess fluid. 7. Place on the positive electrode contact of the gel. NOTE: The wicks must lie parallel to each other on the ends of the gel, evenly touching the surface. 8. Place a glass plate on top of the gel and wicks for approximately one minute. NOTE: This ensures uniformity of contact between wicks and gel and serves to smooth the wick surface in preparation for electrode contact. Materials Horizontal IEF chamber Fine-grained blotting paper Razor blade Refrigerated circulator bath set to 10 C - 15 C Kimwipe Tissues or equivalent Electrode wicks Scissors Forceps Glass plate slightly larger than the gel Sample applicator mask Power supply Reagents Distilled water Catholyte solution Anolyte solution Procedure for sample application 1. Place the sample applicator mask across the gel at least 1 cm from either wick (e.g., 3 cm from cathode). 2. Load sample and pi markers into the slots (2 μl - 5 μl maximum; 2-10 mg/ml concentration). NOTE: In direct tissue isofocusing, tissue samples may be placed directly onto the applicator slots. 3. Ensure electrodes and electrical contacts are clean and there are no breaks in the wire or ribbon. 4. Place the electrodes on the wicks (not the gel surface), aligning them so they lie in parallel upon the wicks. 5. Set the power supply at 1 W constant power. 6. Apply power for 10 minutes. 7. Turn power off. 8. Remove the sample applicator mask. 9. Gently remove any precipitated sample from the gel surface with blotting paper. 182

191 Section XII: Isoelectric Focusing of Proteins on Agarose Gels Running Agarose IEF Gels continued IEF power settings and focusing time 1. Start circulation of the coolant to the IEF chamber. 2. Set the voltage, current and power according to the appropriate running conditions listed in the table below. 3. Once focusing is complete, turn off the power. 4. Discard the wicks. 5. Place the gel in fixative solution. NOTE: The separation progress can be monitored by observing the visible proteins in the pi markers coming into focus and noting the decreasing rate of current flow on the power supply s milliampere indicator. Focusing is attained when the visible pi markers are sharply resolved and the current has stopped decreasing significantly (less than 1 ma in 10 minutes). Running Conditions Ampholyte Voltage Current Power Anolyte Catholyte Focusing Time ph Range (limiting) (upper limiting) (minutes) V MAX 10 W *1 M H 3 PO M Threonine V MAX 10 W 0.5 M HOAc 1 M NaOH V MAX 25 W 0.5 M HOAc 1 M NaOH V MAX 25 W 0.5 M HOAc 1 M NaOH V MAX 10 W 2% solution 0.04 M L- of ph Histidine Ampholyte (free-base) V MAX 10 W 2% solution 0.1 M NaOH or of ph M Bicine Ampholyte 90 *1M H 3 PO 4 can be replaced by 0.5 M acetic acid Isoelectric Focusing of Proteins on Agarose Gels 183

192 Section XII: Isoelectric Focusing of Proteins on Agarose Gels Staining Proteins in Agarose IEF Gels Isoelectric Focusing of Proteins on Agarose Gels Introduction Either Coomassie Blue or Crowle s Double Stain can be used to stain IEF gels. Coomassie stain is used when increased sensitivity is desired, and Crowle s stain produces gels with clear background and sharp resolution. Staining proteins with Coomassie Blue Stain or Crowle s Double Stain Preparation of staining solutions Fixative solution 180 ml Methanol 30.0 g Ttrichloroacetic acid 18.0 g Sulfosalicylic acid Adjust volume to 500 ml with distilled water Coomassie Stain 1.0 g Coomassie Brilliant Blue R ml Ethanol 90 ml Glacial acetic acid Adjust volume to 1 liter with distilled water Coomassie Destaining Solution 250 ml Ethanol 90 ml Glacial acetic acid Adjust volume to 1 liter with distilled water Crowle s Double Stain 2.5 g Crocein scarlet (C.I ) mg Coomassie Brilliant Blue R ml Glacial acetic acid 30.0 g Trichloroacetic acid Adjust volume to 1 liter with distilled water Crowle s Destaining Solution 3 ml Glacial acetic acid Adjust volume to 1 liter with distilled water Materials Flask Stir bar Magnetic stir plate Forceps Paper towel Whatman 3MM chromatography paper 1 kg-2 kg weight Glass plate Forced hot air oven set to 50 C-55ºC Staining vessel Clamps Reagents Methanol Trichloroacetic acid Sulfosalicylic acid Distilled water Coomassie Brilliant Blue R-250 Ethanol Glacial acetic acid Crocein scarlet (C.I ) 184

193 Section XII: Isoelectric Focusing of Proteins on Agarose Gels Staining Proteins in Agarose IEF Gels Continued Follow the steps below to stain the gel after electrophoresis using either Coomassie Blue or Crowle s Double Dtain. 1. Place the gel in Fixative solution for 10 minutes. 2. Remove the gel and rinse the surface with distilled water. 3. Drain excess water. 4. Place on a paper towel, gel side up. 5. Pre-wet a piece of Whatman 3MM chromatography paper with distilled water. 6. Place on gel surface. 7. Overlay the blotting paper with four to six layers of absorbent paper toweling. 8. Place a glass plate on top of the paper toweling. 9. Place a 1 kg - 2 kg weight on top of the toweling for 10 minutes. 10. Remove the weight, the glass plate, and the paper toweling. 11. Rewet the blotting paper thoroughly with distilled water. 12. Gently lift the blotting paper off gel surface. 13. Wash the gel in distilled water for 5 minutes to remove residual fixative and ampholytes. 14. Dry the gel completely in a forced hot air oven (50 C - 55ºC). NOTE: Drying usually takes less than 30 minutes. 15. Stain with Coomassie or Crowle s double stain solution for minutes without agitation. NOTE: Float gel-face down into the stain, so precipitated stain will not settle on the gel surface. 16. Remove the gel and rinse with distilled water. 17. Place the gel in Destaining solution for 3 minutes. 18. Briefly rinse again in distilled water. 19. Clamp (gel side out), onto a glass plate to prevent curling during drying. 20. Dry the gel in a forced hot air oven (50 C - 55ºC) for approximately 15 minutes or dry at room temperature overnight. NOTE: Gel may crack if over dried by heating. Isoelectric Focusing of Proteins on Agarose Gels 185

194 Section XII: Isoelectric Focusing of Proteins on Agarose Gels Staining Proteins in Agarose IEF Gels continued Isoelectric Focusing of Proteins on Agarose Gels Staining Proteins in Agarose IEF Gels with Silver Stain A modified silver stain procedure has been developed for use with agarose gels cast on GelBond Film. After electrophoresis, the gels are fixed, press blotted, and completely dried before staining. Perform all fixing and staining steps in acid-cleaned (50% HNO 3 ) glassware. All washes are done with constant agitation in a volume of at least 250 ml (gel volume:reagent volume = 1:22). Coomassie Brilliant Blue stained gels may be silver stained after drying. In this case, proceed directly to step 14. Preparation of staining solutions Fixative solution 180 ml Methanol 30.0 g Trichloroacetic acid 18.0 g Sulfosalicylic acid Adjust volume to 500 ml with distilled water Silver Stain Solution A 50.0 g Sodium carbonate, anhydrous in 1 liter distilled water (Stable for 2-3 weeks at room temperature) Silver Stain Solution B In the order given dissolve the following reagents in 1 liter of distilled water, while mixing rapidly. 2.0 g Ammonium nitrate 2.0 g Silver nitrate 10.0 g Dodeca-tungstosilicic acid 6.7 ml 37% formaldehyde (Stable 1 week at room temperature stored in the dark) Stop solution 1% Acetic acid Materials Paper towel Whatman 3MM chromatography paper 1 kg-2 kg weight Glass plate Forceps Clamps Forced hot air oven set to 50 C-55 C Staining containers Beakers Magnetic stir plate Magnetic stir bar Acid-cleaned dish Orbital or rocking platform shaker Reagents Methanol Trichloroacetic acid Sulfosalicylic acid Distilled water Glutaraldehyde Dithiothreitol (DTT) Anhydrous sodium carbonate Ammonium nitrate Silver nitrate Dodeca-tungstosilicic acid (Gallard-Schlesinger Cat. No ) 37% formaldehyde Acetic acid Caution: Materials and methods shown here present hazards to the user and the environment. Refer to the safety information on page 210 before beginning these procedures. Caution: Acetic acid causes burns and respiratory irritation. Take precautions to prevent expursure. Caution: Glutaralhyde is toxic and must be handled in a fume hood. 186

195 Section XII: Isoelectric Focusing of Proteins on Agarose Gels Staining Proteins in Agarose IEF Gels continued Follow the steps below to stain the gel after electrophoresis using silver stain 1. Place the gel in Fixative solution for 10 minutes. If gel is prestained with Coomassie Blue and dried, proceed to step Place the gel on a paper towel, gel side up. 3. Pre-wet a sheet of Whatman 3MM chromatography paper with distilled water. 4. Place on gel surface. 5. Overlay the blotting paper with four to six layers of absorbent paper toweling. 6. Place a glass plate on top of the paper toweling. 7. Place a 1 kg - 2 kg weight on top of the toweling for 10 minutes. 8. Remove the weight, the glass plate, and the paper toweling. 9. Rewet the blotting paper thoroughly with distilled water. 10. Gently lift the blotting paper off the gel surface. 11. Wash the gel in distilled water for 5 minutes to remove residual fixative and ampholytes. 12. Clamp (gel side out), onto a glass plate to prevent curling during drying. 13. Dry the gel in a forced hot air oven (50 C - 55ºC) for approximately 15 minutes or until dry. NOTE: Gel may crack if over dried by heating. 14. Soak the dried gel in 2% glutaraldehyde for 10 minutes. 15. Wash in distilled water for 10 minutes using mild agitation. 16. Soak the gel for 10 minutes in 0.01% (DTT) dithiothreitol. 17. Wash in distilled water for 10 minutes using mild agitation. 18. Pour an equal volume of Silver stain solution B into vigorously stirring Silver stain solution A (75 ml B and 75 ml A for each gel to be stained). 19. Transfer the solution to an acid-cleaned glass dish containing one gel. 20. Stain the gel for 10 minutes with gentle agitation. 22. Rinse the gel in distilled water. 23. Wipe any silver deposits from the back of the film. 24. Allow to air dry. Isoelectric Focusing of Proteins on Agarose Gels NOTE: There will be some background. 21. Transfer the gel to a Stop solution and gently agitate for 5 minutes. 187

196 Section XII: Isoelectric Focusing of Proteins on Agarose Gels Press Blot Transfer of Proteins from Agarose IEF Gels Isoelectric Focusing of Proteins on Agarose Gels Press blot transfer is a quick method of removing proteins from agarose gels. The procedure involves overlaying the gel with a buffer-soaked nitrocellulose membrane covered by a thick filter pad and several layers of dry paper toweling. The assembly is then covered by a glass plate. After just 1 1 / 2 minutes of press blot, approximately 20% of the proteins are transferred from the gel to the membrane. Up to 85% transfer can be achieved with a minute blotting time. Transferred proteins can be detected on the membrane and on the gel by standard methods. Procedure 1. Prepare Tris-saline buffer, ph Cut one piece of nitrocellulose membrane and thick filter paper to the same dimension as the gel. NOTE: Wear gloves to prevent contamination by extraneous proteins. 3. Evenly wet the nitrocellulose membrane in the Trissaline buffer by holding one end of the membrane with smooth-tipped forceps and lowering the other end into the buffer container, dropping the membrane flat on the buffer surface. NOTE: The membrane must be completely saturated with buffer. 4. Remove the gel from the focusing chamber. 5. Place on a flat surface, gel side up. 6. Place the buffer-soaked nitrocellulose membrane onto the gel surface. NOTE: Avoid trapping air bubbles between the gel and the membrane. 7. Place one piece of buffer soaked filter paper on top of the membrane. 8. Place three layers of dry paper toweling on top of the filter paper. 9. Cover with a glass plate slightly larger than the gel surface. No other weight is required. 10. Press blot for 1 1 /2 minutes or longer, as desired. 11. Remove glass plate and discard the paper toweling and filter paper. Materials Nitrocellulose membrane Thick filter paper Scissors Smooth-tipped forceps Container Paper towels Glass plate Reagents Tris-saline buffer, ph 7.5 (50.0 g Tris-HCl, 0.94 g Tris-Base, g NaCl adjust to 2 liters with distilled water) 188

197 Section XII: Isoelectric Focusing of Proteins on Agarose Gels Preparative Isoelectric Focusing Separation of relatively large amounts of biologically active macromolecules is possible by isoelectric focusing in agarose. Typical high-yield recoveries of applied proteins are obtainable with retention of biological activity. This preparative isoelectric focusing procedure is based on the work of Cantarow, et al. As much as mg of protein can be focused in 9.5 ml (0.75 x 10 x 11.5 cm) of 1% IsoGel Agarose containing 2.5% ampholytes. Procedure 1. Follow steps 1-14 for Gel Preparation on pages Also see Sample Preparation on page Add the protein sample to the agarose solution once cooled to 60ºC. 3. Stir the solution well to mix. 4. Cast the gel and disassemble the casting cassette following the steps previously described on page Place the gel on the 10º C cooling platen for 10 minutes (if gel has not already been chilled after casting). 6. Focus following the steps previously described on pages Procedure for recovering proteins from preparative gel 1. Excise the gel slice containing the protein of interest by using a spatula to strip the agarose from the GelBond Film in 5 mm-wide slices. 2. Place the agarose strip in a 5-cc plastic syringe fitted with an 18-gauge needle. 3. Macerate the gel slice by expelling it into a clean tube. 4. Add 4 ml of phosphate-buffered saline (PBS) to the macerated gel. 5. Cover the tube securely with Parafilm. 6. Place on a test tube rocker for 16 hours at 4ºC. 7. Centrifuge the tube for one minute at 100 rpm. 8. Separate the supernatant from the gel using a serum separator. Materials Spatula 5-cc plastic syringe with an 18-gauge needle Clean centrifuge tube Test tube rocker at 4 C Centrifuge Serum separator Parafilm Reagent Phosphate-buffered saline (PBS) Isoelectric Focusing of Proteins on Agarose Gels 189

198 Section XII: Isoelectric Focusing of Proteins on Agarose Gels Immunofixation/Immunoperoxidase or Autoradiography Isoelectric Focusing of Proteins on Agarose Gels Detection of separate species can be accomplished by protein stains or by overlaying gels with specific antibody solutions coupled to enzymes that will eventually produce a visible end product. The antibodyperoxidase conjugate system or autoradiography with 125 I- labeled antibody are frequently used for this purpose. Immunoperoxidase labeling of focused proteins is performed according to Saravis, et al. After focusing, fixing, and drying the gel, treat the gel as described below. Procedure for Immunoperoxidase Staining/Avidin-Biotin Modification 1. Soak the gel in 3% hydrogen peroxide for 10 minutes. 2. Rinse with distilled water. 3. Soak the gel in 2.28% periodic acid for 5 minutes. 4. Rinse with distilled water. 5. Soak the gel in 0.02% sodium borohydride for 2 minutes. 6. Rinse with distilled water. 7. Place the gel in 0.05 M Tris-saline, ph 7.6 for 10 minutes. 8. Incubate the gel with 1:5 normal serum for 10 minutes (same species as secondary antibody). 9. Incubate the gel for 2-4 hours at room temperature with Anti-serum(primary antibody). 10. Rinse the gel with 0.15 M PBS, ph 7.4 for 1 hour at room temperature. 11. Incubate the gel with Secondary* antibody (e.g., goat anti-mouse IgG) for 2-4 hours at room temperature. NOTE: If the avidin/biotin modification is used, proceed with steps 12 and 13 using biotinylated reagents (marked with * in steps 11 and 15). 12. Rinse the gel with 0.15 M PBS for 1 hour at room temperature. 13. Incubate the gel with avidin solution (40 mg/gel) in PBS for 1 hour at room temperature (stock solution of avidin is usually 20 mg/ml). 14. Rinse the gel with 0.15 M PBS for 1 hour at room temperature. 15. Incubate the gel with horseradish peroxidase* (33 mg/ gel) for 2 hours at room temperature. 16. Rinse the gel with 0.15 M PBS for 1 hour at room temperature. 17. Incubate the gel with diaminobenzidine (0.15 mg/ml) and hydrogen peroxide (0.03%) in 0.01 M Tris 0.15 M NaCl for 1-17 hours at room temperature. 18. Rinse the gel in PBS and dry. Materials Staining containers Reagents Hydrogen peroxide Distilled water Periodic acid Sodium borohydride 0.05 M Tris-saline (ph 7.6), normal serum (same species as secondary antibody) Anti-serum (primary antibody) 0.15 M PBS (ph 7.4) Secondary antibody 20 mg/ml avidin solution Horseradish-peroxidase 0.15 mg/ml diaminobenzidine 0.01 M Tris/0.15 M NaCl 190

199 Section XII: Isoelectric Focusing of Proteins on Agarose Gels Direct Tissue Isoelectric Focusing (DTIF) This method employs the application of tissue or cell pellets directly onto the surface of the IEF gel without concentration of samples, dialysis to remove salts, or the salt extraction of soluble proteins from tissue. DTIF allows more soluble protein per milligram of tissue to enter the gel than is recoverable by extraction procedures and minimizes denaturation of biologically active proteins that can be damaged by extraction. 1. Prepare solutions and cast gels as previously described on pages Prepare for focusing as previously described on page Place the sample applicator mask across the gel at least 1 cm from either wick (e.g., 3 cm from cathode). 4. Drape a tissue slice over the open slot of the applicator mask. 5. Ensure the electrodes and the electrical contacts are clean and there are no breaks in the wire or ribbon. 6. Place the electrodes on the wicks (not the gel surface), aligning them so they are in parallel upon the wicks. 7. Set power supply at 1 W (constant power). 8. Prefocus for minutes to allow sample uptake. 9. Turn power supply off. 10. Remove the tissue slice and the applicator mask. 11. Focusing is continued using standard conditions. 12. After focusing is complete, the gel is fixed, stained and dried following standard procedures. Isoelectric Focusing of Proteins on Agarose Gels 191

200 Section XII: Isoelectric Focusing of Proteins on Agarose Gels Resolution Reference Guide Phenomenon IEF Band Appearance Possible Causes Remedy Isoelectric Focusing of Proteins on Agarose Gels Streaks or gaps perpendicular to bands applied too close to pl point applying sample. Zwitterionic Fuzzy bands gradient before removing gel from platen. fixative immediately after IEF run. Skewed bands gel parallel to each other. uid is squeezed from wicks. > 5 mm from edge of gel. dialyze against 1% glycine. Missing or faint bands denaturing conditions. of application Wavy bands dialyze against 1% glycine. distorting ph gradient Arc-shaped bands ready for use. handling gel. 192

201 Section XII: Isoelectric Focusing of Proteins on Agarose Gels Resolution Reference Guide continued Phenomenon IEF Band Appearance Possible Causes Remedy Burning or sparking cathodal wick should be damp. to obtain linear ph gradient. Sparking along edge of gel onto cooling plate Formation of ditch ultimately causing gel to burn out EEO fiow of water to cathode on gel early in run blotting gel. run during sample uptake. before use. moisture. Sample smearing or precipitation or 1% glycine. insoluble detergents may be necessary. Incomplete Sample Uptake Sample retained in sample application well Fluid expression damp before application. blotted before use. solution onto gel electrode wicks. Persists for duration of run saturating wick) cathrode; higher at anode) gel film and chamber platen power supply. Reverse polarity if incorrect. Isoelectric Focusing of Proteins on Agarose Gels 193

202 Section XII: Isoelectric Focusing of Proteins on Agarose Gels Resolution Reference Guide continued Isoelectric Focusing of Proteins on Agarose Gels Phenomenon IEF Band Appearance Possible Causes Remedy Condensation inside chamber thereafter (ph 3-10 e.g.). tray in chamber. Get thinning Uneven ph gradient/ph gradient different than stated for ampholyte used References humidity conditions electrolyte solutions place in humidity chamber. linear ph gradient. contaminants. ph gradient is not linear Measure ph gradient before removing gel. ph gradient not attaining upper limits ph range shifted toward cathode towards cathode) attain desired gradient profile. Current increases with time ends of the gel solution. electrode to cathode. Diagonal band migration perpendicular to the sample migration. Lateral band migration applied. concentration. Sample focuses at wrong position characteristic. A supposedly pure sample focuses as multiple bands High background stain procedure. destaining. solution. minutes. Delamination (peeling) of gel from film support on staining after drying procedure. 194 Allen, R.C., et al., Gel Electrophoresis and Isoelectric Focusing of Proteins: Selected Techniques, Walter de Gruyter & Co., Westermeier, R., Electrophoresis in Practice, 2nd edition, Wiley Company, Coligan, J.E., et al., Current Protocols in Protein Science, Wiley Company, Harper, D.L., Electrophoresis 81, , Saravis, C.A. and Zamcheck, N., J. Immunol.Methods, 29: 91-96, Cantarow, W.D., et al., Electrophoresis, 3: 85-89, Ebers, G.C., et al., J. Immunol. Methods, 37: , Saravis, C.A., et al., Electrophoresis, 1: , Saravis, C.A., et al., J. Immunol. Methods, 29: , 1979.

203 Section XIII: Protein Separation in Agarose Gels Protein Separation in Agarose Gels In This Section Introduction 196 Buffers for Protein Separation in Agarose 197 Casting Agarose Gels for Protein Separation 198 Preparation and Loading of Protein Samples 198 Optimal Voltage and Electrophoretic Times 199 Detection of Proteins in Agarose Gels 199 Gel Drying and Preservation 200 Processing Agarose Gels Following Electrophoresis 200 Recovering Proteins from Agarose Gels 201 References

204 Section XIII: Protein Separation in Agarose Gels Introduction Protein electrophoresis in agarose gels is an alternative approach to using polyacrylamide gels and provides several benefits. Gels can be run using a vertical system or a horizontal system and unlike polyacrylamide gels, agarose gels can be used effectively to separate proteins larger than 600,000 Da. Advantages Separate high-molecular-weight proteins (>600,000 Da) Easy to prepare and handle Efficient recovery of proteins Excised proteins can be used to immunize animals directly for antibody production Non-toxic Recommended agaroses for protein electrophoresis The table below is a list of Lonza Agaroses that are recommended for protein electrophoresis. For performing routine separations, we recommend a standard melting temperature agarose such as MetaPhor Agarose or SeaKem Gold Agarose. When proteins are to be recovered for further analysis, use a low melting temperature agarose such as SeaPlaque GTG or NuSieve GTG Agarose. Protein Size Range(kDa) Agarose Concentration MetaPhor or NuSieve GTG 5% MetaPhor or NuSieve GTG 3% MetaPhor or NuSieve GTG 2% 600-1,000 SeaKem Gold or SeaPlaque 1.5% 1,000-5,000 SeaKem Gold or SeaPlaque 1.0% Protein Separation in Agarose Gels 196

205 Section XIII: Protein Separation in Agarose Gels Buffers for Protein Separation in Agarose The buffer systems used for agarose electrophoresis are similar to those used for polyacrylamide electrophoresis. When performing horizontal electrophoresis, we have found that a Tris-borate gel and running buffer provide greater resolution than using the standard Laemmli Buffer system. Vertical and horizontal gel 1X Stack Buffer, ph 8.0 g/l for 1X Stack Buffer 125 mm Tris-HCl 19.7 g Tris-HCl in 1 liter distilled water NOTE: Horizontal gels do not require the use of a stack gel. 1X Resolving Buffer, ph 8.5 g/l for 1X Resolving Buffer 500 mm Tris base g Tris base 160 mm Boric acid 9.90 g Boric acid 1 M Urea g Urea Adjust volume to 1 liter with distilled water 1X Running Buffer, ph 8.5 amount for 1X Running Buffer 90 mm Tris base g Tris base 90 mm Boric acid 5.57 g Boric acid 0.1% SDS 10 ml of 10% SDS Adjust volume to 1 liter with distilled water Laemmli Buffer Gels can be made with standard Laemmli Buffer (see page 156). To avoid excess foaming during agarose dissolution; only add SDS to the cathodal buffer and not to the gel buffer. The SDS in the cathodal buffer will migrate faster than the proteins during electrophoresis, maintaining protein denaturation. To prevent buffer depletion in vertical systems, use 10X Laemmli Buffer without SDS in the anodal buffer. Tips for buffer preparation Add SDS to the cathodal buffer only. Do not add SDS to the gel prior to dissolution. If other buffer systems are used, the ph should be between ph 5-9. Denaturants such as urea and formamide should only be added at low concentrations (4 M-6 M Urea). For buffers more alkaline than ph 9, dissolve and cast the agarose in distilled water, allow to gel. Soak horizontal gels in the alkaline gel buffer for 30 minutes prior to electrophoresis. For vertical gel systems, prerun the gel to equilibrate the gel with the alkaline buffer. Caution: Materials and methods shown here present hazards to the user and the environment. Refer to the safety information on page 210 before beginning these procedures. Protein Separation in Agarose Gels 197

206 Section XIII: Protein Separation in Agarose Gels Casting Agarose Gels for Protein Separation Protein Separation in Agarose Gels The procedures for dissolving and casting agarose gels for protein separation are the same as the procedures used for nucleic acid separation. Refer to Dissolving Agarose and Casting Agarose Gels (see Section II). Tips for casting horizontal agarose gels Use of a stacking gel is not necessary for horizontal submarine electrophoresis. The resolving gel buffer and running buffer should be the same. Dissolve the agarose in running buffer without SDS. For denaturing electrophoresis, add SDS to the sample buffer and the running buffer. Let the gel set for minutes at room temperature. For MetaPhor and SeaPlaque Agarose, chill the gel at 4 C for minutes before removing comb. If gels are to be dried, cast the gels onto GelBond Film. Tips for casting vertical agarose gels Stacking gel for vertical gels Prepare a 1% SeaKem Gold Agarose gel in stacking gel buffer. For proteins >100 kda, the use of a stacking gel may be omitted. It will not enhance band resolution. After the stacking gel is set, place cassette at 4 C for 30 minutes prior to removing the comb. Resolving gel for vertical gels Dissolve the agarose in running buffer without SDS. Refer to Vertical Gel Casting Instructions (see page 86). Allow resolving gel to set approximately 3 minutes at room temperature then cast the stacking gel. To facilitate comb removal from a vertical gel The teeth of the comb can be tapered so the width at the bottom is slightly smaller than at the top. A slight rounding of the edges is all that is needed so that the end is U-shaped. Tapering the teeth in this way will not affect the pattern of the protein bands. Flood the comb area with running buffer prior to removing the comb. If clamps are used, remove the clamps at the top of the gel cassette and gently loosen the comb by moving it forward and back before removal. Preparation and Loading of Protein Samples Sample preparation and amount of protein that can be loaded on agarose gels is essentially the same as for polyacrylamide gels and is largely dependent on your application and detection method. Guidelines Suspend protein samples in 2X sample buffer, 1:1 (v:v). If denatured proteins are required, incubate at 95 C-100 C for 5 minutes. Load the samples into the sample wells. The minimal amount of protein detectable by Coomassie Brilliant Blue stain is about 1.0 μg; and may vary depending on the protein. Larger amounts of protein can be loaded, but band thickness increases accordingly. For a 0.8 cm wide well, 25 ml (50 μg total protein) is recommended for a complex mixture, if staining with Coomassie Blue, and 1 ml (10 μg total protein) is needed for samples containing one or a few proteins. For vertical electrophoresis, load empty wells with sample buffer. 2X Tris-Glycine SDS sample buffer for agarose electrophoresis of proteins 2X concentrate Amount to add for 10 ml 126 mm Tris-HCl, ph ml of 0.5 M Tris-HCl, ph % Ficoll Type g Ficoll Type 400 4% SDS 4 ml of 10% SDS 0.002% Bromophenol Blue 0.2 ml of 0.1% Bromophenol blue Adjust volume to 10 ml with distilled water Before use: add 1 ml -Mercaptoethanol ( ME) to 10 ml of 2X Tris-Glycine SDS sample buffer 198

207 Section XIII: Protein Separation in Agarose Gels Optimal Voltage and Electrophoretic Times Tips Avoid higher power settings as the heat generated may melt the agarose. Thick vertical gels (>1 mm) will require proportionally higher current settings to complete the electrophoresis run within the times indicated. Electrophorese the gel until the tracking dye travels to the bottom of the resolving gel. Prestained molecular weight markers such as Lonza s ProSieve Color Protein Marker can be used to monitor electrophoresis. The gels can be electrophoresed longer, but care should be taken that smaller proteins do not travel off the gel. Gel Type Gel Size Power Setting Time Horizontal Gel Vertical Gel Mini-Vertical 5.3 cm x 8.5 cm x.4 cm 14.5 cm x 16.5 cm x.1 cm 8 cm x 10 cm x.1 cm 100 volts 25 ma (constant) 20mA (approximately 80 volts at the (approximately 50 volts at the start and 200 volts at the end) start and 120 volts at the end) 3-4 hours hours hours Section XIV: Protein Separation in Agarose Gels Detection of Proteins in Agarose Gels The procedures for staining agarose gels with Coomassie Blue Stain are essentially the same as they are for polyacrylamide gels with some modifications (listed below). For detailed procedures on Coomassie Brilliant Blue staining (see page 168), using the modifications listed below. For detailed procedures on Silver staining, refer to Staining Proteins with Silver Stain (see page 186 in Isoelectric Focusing of Proteins on Agarose Gels). Tips Agarose gels require more time to process than polyacrylamide gels of similar dimensions. Staining and destaining times will vary depending on the gel concentration, thickness and protein concentration. Place container on shaker with gentle motion during staining and destaining procedures. Staining proteins with Coomassie Brilliant Blue Stain Coomassie Blue Stain solution Destain Solution 40% Methanol 20% Methanol 10% Glacial Acetic Acid 5% Glacial Acetic Acid 0.25% Coomassie Brilliant Blue R-250 Room temperature staining A 14.5 cm x 16.5 cm, 1 mm thick agarose gel will stain in approximately 1 to 2 hours. Destain for 1-4 hours with gentle shaking at room temperature. Overnight staining Use 0.125% Coomassie Blue R-250 with the same concentrations of methanol and acetic acid as in the stain solution. Destain approximately 4 hours. Accelerated staining Stain gels using standard stain solutions at 50 C. A 1 mm thick gel takes approximately 1 hour to stain and 1 hour to destain. Change the destaining solution 1 time. Agarose gels become softer at 50 C - use a support to transfer between solutions. Storage Do not store agarose gels in destain solution, they may become brittle and fracture. Store gels in a 5% glycerol solution or dried. Protein Separation in Agarose Gels 199

208 Section XIII: Proteins Separation in Agarose Gels Gel Drying and Preservation Agarose gels can be dried overnight at room temperature, dried in a forced hot-air oven or dried using a standard vacuum gel dryer. When not using a vacuum gel dryer, the gel must first have been cast onto GelBond Film to prevent the gel from shrinking during the drying process. The procedures for drying protein agarose gels are the same as drying DNA agarose gels. Proteins Separation in Agarose Gels Processing Agarose Gels Following Electrophoresis Protein Separation in Agarose Gels Autoradiography After drying, agarose gels can be exposed directly to X-ray film. Fluorography Do not immerse agarose gels into any uorography solution if the gels are attached to GelBond Film. Solutions containing high concentrations (>50%) of DMSO must not be used, as they will dissolve the agarose. Commercially prepared solutions which precipitate the uor within the agarose gel matrix (e.g., EN 3 HANCE from Perkin Elmer) work best. Follow the manufacturer s instructions. The uor-impregnated gel can then be dried onto filter paper under vacuum at <50 C in a slab-gel dryer and then exposed directly to X-ray film. Electroblotting proteins from agarose gels Proteins can be electroblotted out of agarose gels onto membranes (nitrocellulose, PVDF, etc.) by using the same methods used for polyacrylamide gels. Refer to Blotting Proteins from Polyacrylamide Gels for detailed procedures (see page 171). It is important to note that agarose gels adhered to GelBond Film cannot be electroblotted because GelBond Film is nonporous. The time required for optimal transfer of specific proteins will need to be determined experimentally. In general, proteins transfer 15% faster out of agarose gels than from a polyacrylamide gel. 200

209 Section XIII: Proteins Separation in Agarose Gels Recovering Proteins from Agarose Gels Proteins can be readily recovered from agarose gels. When protein is to be recovered, the use of a low melting temperature agarose such as NuSieve GTG, MetaPhor or SeaPlaque Agarose is recommended. Protein Recovery Tips Identifying the protein to be recovered from the gel can be accomplished by several methods. If the gel is fixed and stained with Coomassie Blue as detailed earlier, then 1% SDS should be added to the dilution buffer. Recovery of proteins in the native state requires that they not be fixed prior to recovery. Methods for detecting proteins which do not require fixing include: Using prestained molecular weight markers as a guide for the relative position of the desired protein in the gel. Performing a short (<10 minutes) pressure/ capillary blot of the gel so that small (<5%) amounts of the proteins are transferred to a membrane and then gold stained. If prestained molecular weight markers are used, it is possible to place the stained membrane under the gel and identify the region of interest. We recommend the use of 50 mm Tris-HCl, 1 mm EDTA at ph 8.0 as the extraction/dilution buffer. The buffer may require modification depending upon the particular protein to be recovered and what further work is planned after it is recovered. The buffer should be one in which the protein of interest will be stable. The amount of extraction/dilution buffer added is directly related to the amount of protein that can be recovered: the more the agarose is diluted, the more protein will be recovered. Greater than 90% of a protein can be recovered with a dilution to 0.5% agarose. A second dilute-freeze-spin cycle can be performed to recover additional protein. Materials Spatula 1.5 ml microfuge tube(s) Heat block -70 C freezer Microcentrifuge at 4 C Reagents Extraction buffer (50 mm Tris-HCl, 1 mm EDTA at ph 8.0) Ice Protein Separation in Agarose Gels 201

210 Section XIII: Proteins Separation in Agarose Gels Recovering Proteins from Agarose Gels continued Procedure Protein Separation in Agarose Gels 1. Identify the region of the gel which contains the protein to be recovered. 2. Excise a gel slice containing the protein of interest. 3. Place the gel slice in a 1.5 ml microfuge tube. 4. Determine the volume of the gel slice by weight or size. 5. Add the appropriate amount of extraction buffer so the final concentration of agarose is 0.5%. 6. Melt the gel slice by heating to 70 C for SeaPlaque or NuSieve GTG Agarose or 80 C for MetaPhor Agarose. 7. Mix thoroughly. NOTE: Ensure the gel is completely melted and diluted by the buffer. 8. Incubate on ice for 30 minutes. NOTE: It is important that the agarose mixture has gelled as much as possible at this step. 9. Freeze the mixture at -70 C for 1 to 2 hours. 10. Allow the mixture to thaw on ice. 11. Centrifuge the mixture at 13,000 rpm in a microcentrifuge for 10 to 20 minutes at 4 C. 12. Remove the supernatant; this contains the recovered protein. NOTE: Each step of this procedure should be carefully followed to obtain quantitative results. Care should be taken to COMPLETELY gel the protein-agarose solution. If the procedure cannot be completed at one time, we recommend keeping the mixture at -70 C, as prolonged freezing will not affect the protein. Protein recovered from MetaPhor Agarose by this technique will co-purify with some residual agarose. The amount of agarose present is between 1% and 3% of the original amount of agarose present in the gel slice. All of the residual agarose will pass through a 0.1 mm pore filter, about 83% will pass through a 100 kda molecular-weight cutoff filter, and 50% will pass through a 30 kda molecular-weight cutoff filter. The recovered protein can be separated from the residual agarose by conventional chromatography. References 202 FMC BioProducts RESOLUTIONS, 8(1): 6-7, Atkins, T., Am. Biol. Teacher, 53: , Litz, J.S., FMC BioProducts RESOLUTIONS, 6(3): 6, Sakakibara, R., et al., Anal. Biochem., 162: , Morgan, J. H., et al., BioTechniques, 11: , Coligan, J.E., et al., Current Protocols in Protein Science, Wiley & Sons, Inc., Kim, R., et al., Analytical Biochem., 282(1): , Wu, M. and Kusukawa, N., BioTechniques, 24: , 1998.

211 Appendix A: Electrophoretic Theory In This Section Voltage, Current and Power; Interative Effects on Gel Electrophoresis 204 Electrophoretic Parameters 205 Electrophoretic Theory 203

212 Appendix A: Electrophoretic Theory Voltage, Current and Power: Interactive Effects on Gel Electrophoresis Introduction Electrophoresis is defined as the movement of ions and charged macromolecules through a medium when an electric current is applied. Agarose and polyacrylamide are the primary stabilizing media used in the electrophoresis of macromolecules. Macromolecules are separated through the matrix based on size, charge distribution and structure. Proteins will separate through the matrix based on size, structure and charge. In general, nucleic acids migrate through a gel based on size, with little in uence from base composition or sequence. Two equations are relevant to the use of power supplies for electrophoresis of macromolecules: Ohm s Law and the Second Law of electrophoresis. These two laws and the interactions of these parameters (watts, volts, current) are critical to understanding electrophoresis. Ohm s Law Current (I)=Voltage (V)/Resistance (R) Ohm s Law states that current is directly proportional to the voltage and is inversely proportional to the resistance. Resistance of the system is determined by the buffers used, the type and configurations of the gels being run, and the total volume of all the gels being run. Second Law Watts (W)=Current (I) x Voltage (V) The Second Law states that power or watts (a measure of the heat produced) is equal to the product of the current and voltage. Since V=I x R, this can also be written as Watts=I 2 x R. Electrophoretic Theory 204

213 Appendix A: Electrophoretic Theory Electrophoretic Parameters During electrophoresis, one of the parameters is held constant and the other two are allowed to vary as the resistance of the electrophoretic system changes. In vertical systems, the resistance of the gel increases as highly conductive ions like Cl are electrophoresed out of the gel. As these ions are removed from the gel, the current is carried by less conductive ions like glycine, borate, acetate, etc. Under normal conditions in horizontal systems, there is little change in resistance. However, with high voltage or extended runs in horizontal systems, resistance can decrease. Introduction There are advantages and disadvantages for setting each of the critical parameters as the limiting factor in electrophoresis. Sequencing gels are usually run at constant wattage to maintain a uniform temperature. Agarose and acrylamide gels for protein and DNA separation are run at constant voltage or constant current. Constant wattage In a vertical system when wattage is held constant, the velocity of the samples will decrease because the current, which is in part carried by the DNA, decreases to compensate for the increase in voltage. The generation of heat will remain uniform. If the current should decrease disproportionately (from a buffer problem, a buffer leak or a hardware problem), the power supply will increase the voltage to compensate. Since voltage and current vary over time at a constant wattage, it is not possible to predict mobility of samples from the calculation of watt-hours. Contant wattage Volts & Watts Time (hours) Reference ma Volts Watts ma 1. Rickwood, D. and Hames, B.D., Gel Electrophoresis of Nucleic Acids: A Practical Approach, IRL Press Limited, Constant current When the current is held constant, the samples will migrate at a constant rate. Voltage and wattage will increase as the resistance increases, resulting in an increase in heat generation during the run. If a break occurs in the system, such as a damaged lead or electrode or a buffer leak, the resistance of the gel will be greatly increased. This will cause a large increase in wattage and voltage resulting in the generation of excessive heat. It is even possible for the system to get hot enough to boil, or start the apparatus to scorch or burn. Constant current Volts & Watts Constant voltage When voltage is set constant, current and wattage will decrease as the resistance increases, resulting in a decrease of heat and DNA migration. Since the heat generated will decrease, the margin of safety will increase over the length of the run. If a problem develops and the resistance increases dramatically, the current and wattage will fall since the voltage cannot increase. Even if the apparatus fails, the worst that is likely to happen is that the resistance will increase so much that the power supply will not be able to compensate, and it will shut off. Volts & Watts Time (hours) Constant voltage Time (hours) ma ma Volts Watts ma Volts Watts ma Electrophoretic Theory 205

214 Notes 206

215 Appendix B: Agarose Physical Chemistry Agarose Physical Chemistry In This Chapter Agarose Physical Chemistry 208 References

216 Appendix B: Agarose Physical Chemistry Agarose Physical Chemistry Agarose is a polysaccharide consisting of 1,3-linked -D-galactopyranose and 1,4-linked 3,6-anhydro- -Lgalactopyranose (Figure 1). This basic agarobiose repeat unit forms long chains with an average molecular mass of 120,000 daltons, representing about 400 agarobiose units (1). There are also charged groups present on the polysaccharide, most notably pyruvate and sulfate. These residues are responsible for many agarose properties, and by careful selection of raw materials, these properties can be controlled to meet specific needs. OH OH O OH Figure 1: Agarobiose Basic repeating unit of agarose. O O OH O O n SO 4 SO 4 Na+ Na+ + SO 4 Figure 3: Illustration of EEO. Anionic groups are fixed to the matrix and cannot move. Corresponding cations can move and sweep toward the cathode with their associated water of hydration. The mechanism for gelation of agarose was first suggested by Rees (4) and later demonstrated by Arnott, et al. (5). It involves a shift from a random coil in solution to a double helix in the initial stages of gelation, and then to bundles of double helices in the final stage (Figure 4). The average pore size varies with concentration and type of agarose, but is typically 100 to 300 nm (6). Gelation Mechanism COO SO Electroendosmosis (EEO) is a functional measure of the number of these sulfate and pyruvate residues present on the agarose polysaccharide (Figure 2). Electroendosmosis (2,3) is a phenomenon that occurs during electrophoresis when the anticonvective medium (the agarose in this case) has a fixed negative charge. In an electric field, the hydrated positive ions associated with the fixed anionic groups in the agarose gel migrate toward the cathode. Water is thus pulled along with the positive ions, and migration of negative molecules such as DNA is retarded (Figure 3). Electroendosmosis is quantitated by subjecting a mixture of dextran and albumin to electrophoresis, then visualizing them and measuring their respective distances from the origin. The amount of EEO (-mr) is calculated by dividing the migration distance of the neutral dextran (OD) by the sum of the migration distances of the dextran and the albumin (OD + OA): -mr = OD/(OD + OA). EEO mr meq SO4 + pyruvate/100g Figure 2: Dependence of electroendosmosis on total anionic residues in agarose. Solution COOL HEAT Gel I COOL HEAT Gel II Figure 4: Gelation of agarose by formation of double helices connected in three dimensions by zones of random coil configuration. One of the most important factors contributing to the success of agarose as an anticonvection medium is its ability to exhibit high gel strength at low concentrations ( 6%). Gel strength is defined as the force, expressed in g/cm 2, that must be applied to fracture an agarose gel of a standard concentration. As there are several test methods used to measure gel strength, a direct comparison of gel strength values between different manufacturers is sometimes difficult. The gel strength of a specific lot of agarose will decrease over time because of the spontaneous hydrolysis of the agarose polysaccharide chains. This loss of gel strength can be particularly noticeable after 5 years from the manufacturing date. Agents that disrupt hydrogen bond formation (chaotropic agents such as urea and potassium iodide) will decrease the melting temperature, gelling temperature and gel strength of agarose gels, or even inhibit the formation of the gel. Since some electrophoretic procedures call for the incorporation of urea into the gel, it is important to be aware of this effect.

217 Appendix B: Agarose Physical Chemistry Continued The energy needed to melt an agarose gel increases as the gel concentration increases. The gelling temperature of an agarose gel is also in uenced by the gel concentration (Figure 5). For this reason, gelling or remelting temperatures are expressed at a given agarose concentration. This property is of practical value since it is possible to vary gelling and melting parameters by using lower or higher concentrations of agarose. The depen dence of gelling and melting temperatures on concentration is most pronounced at concentrations less than 1%. Temperature, C SeaKem LE Agarose SeaPlaque Agarose SeaPrep Agarose gelling temperature. Because these agarose types have a melting temperature of 65 C, it is possible to remelt the agarose without melting the DNA double helix. Thus, slices of SeaPlaque GTG or NuSieve GTG Agarose gels containing DNA can be melted and the nucleic acids manipulated directly in the remelted agarose. Reduction in Gelling Temp., C Hydroxyethyl 15 Hydroxypropyl Methyl 10 Allyl 5 Acetyl Degree of Substitution/Agarobiose Unit (Max. = 4) Figure 7: Effect of various substituents on agarose gelling temperature. Agarose Physical Chemistry Concentration, % Figure 5: Effect of concentration on gelling (open symbols) and melting (solid symbols) temperatures of agaroses. The agarose polysaccharide also contains uncharged methyl groups. The extent of natural methylation is directly propor tional to the gelling temperature. Unexpectedly, synthetically methylated agaroses have lower, rather than higher, gelling temperatures (Figure 6, lower curve), and the degree of synthetic methylation is inversely proportional to the melting temperature. A subsequent study demonstrated that a number of simple derivatives have the same effect as synthetic methylation on gelling temperature (Figure 7). Because of manufacturing concerns, hydroxy ethylation is preferred for large scale derivatization of agarose. The major use of low gelling temperature agarose, such as SeaPlaque GTG and NuSieve GTG Agarose, is to take advantage of the low melting point rather than the low References 1. Rochas, C., and Lahaye, M. (1989) Carbohydrate Polymers 10: Himenz, P.C. (1977) Principles of Colloid and Surface Chemistry, Marcel Decker. 3. Adamson, A.W. (1976) Physical Chemistry of Surfaces, John Wiley & Sons. 4. Rees, D.A. (1972) Biochem. J. 126: Arnott, S., et al. (1974) J. Mol. Biol. 90: Griess, G.A., et al. (1989) Biopolymers 28: Gelling Temperature, C Synthetic Natural % Methoxyl Figure 6: Dependence of agarose gelling temperature on natural and synthetic methylation. 209

218 Appendix C: Safety and Environmental Precautions Safety & Environmental Precautions In general, working with Nucleic Acids and Proteins does not present significant hazards to humans so long as precautions are taken to protect against certain harmful materials. Throughout The Sourcebook are references to materials and methods which are hazardous to humans and the environment. Specific hazards and protection steps are summarized here, and it is recommended that trained users follow these precautions when performing the operations outlined in this manual. These precautions are not a substitute for proper health and safety training, nor are they a substitute for the user institution s standards and procedures, or local governmental requirements. In each case, the user should be aware of and follow local guidelines for handling and disposal of these materials. Specific Chemical Hazards Ethidium Bromide Ethidium bromide (EtBr) is a known mutagen and a suspected carcinogen. Care should be taken to prevent exposure. Recommended personal protective equipment includes: nitrile gloves, lab coats, and safety glasses. Use ethidium bromide solutions in a well ventilated area, and prevent inhalation of vapors. Electrophoresis tanks should be kept covered during electrophoresis of gels containing ethidium bromide. Ethidium bromide powder should be handled in a fume hood. Decontamination and disposal of ethidium bromide should be done according to local government and institutional regulations. GelStar and SYBR Green Nucleic Acid Gel Stains GelStar and SYBR Green Stains contain a component which can penetrate cells (including skin), and is a potential mutagen. Therefore care should be taken to prevent exposure. Recommended personal protective equipment includes: (non nitrile) gloves, lab coats, and safety glasses. Decontamination and disposal of these stains should be done according to local government and institutional regulations. Formaldehyde and Formamide Formaldehyde and Formamide are known carcinogens, and exposure should be limited to as low as feasible. Gloves, safety glasses (or goggles when pouring liquid), and lab coats should be worn. Operations using formaldehyde or formamide should be conducted with the use of respiratory protection, or in a fume hood or other well ventilated area to prevent inhalation. Disposal of these materials should be done according to local government and institutional regulations. DMSO Dimethyl Sulfoxide (DMSO) can penetrate cells and DNA, and is a carrier of other substances in solutions into those cells. Care should be taken to prevent exposure, including the use of non nitrile gloves (natural rubber are recommended), safety glasses and lab coat. Disposal of these materials should be done according to local government and institutional regulations. Phenol Phenol is toxic by inhalation, ingestion, and contact. It will burn eyes. Appropriate gloves, safety glasses (or goggles), and lab coats are required. Proper ventilation should be utilized. Disposal of these materials should be done according to local government and institutional regulations. 210

219 Indices Chapter 4

220 Numerical Index Indices Cat. No. Description Page Number SeaKem LE Agarose 125 g SeaKem LE Agarose 25 g SeaKem LE Agarose 100 g SeaKem LE Agarose 500 g SeaKem LE Agarose 1 kg SeaKem ME Agarose 125 g SeaKem ME Agarose 125 g SeaKem ME Agarose 25 g SeaKem ME Agarose 25 g SeaKem ME Agarose 500 g SeaKem ME Agarose 500 g SeaKem HE Agarose 125 g SeaKem HE Agarose 25 g SeaKem HEEO Agarose 125 g SeaKem HEEO Agarose 25 g SeaKem HGT Agarose 125 g SeaKem HGT Agarose 25 g SeaKem HGT-P Agarose 125 g SeaKem GTG Agarose 125 g SeaKem GTG Agarose 25 g SeaKem GTG Agarose 500 g NuSieve GTG Agarose 125 g NuSieve GTG Agarose 25 g NuSieve GTG Agarose 500 g NuSieve 3:1 Agarose 125 g NuSieve 3:1 Agarose 25 g NuSieve 3:1 Agarose 500 g SeaPlaque Agarose 125 g SeaPlaque Agarose 125 g SeaPlaque Agarose 25 g SeaPlaque Agarose 25 g SeaPlaque GTG Agarose 125 g SeaPlaque GTG Agarose 25 g InCert Agarose 1 g InCert Agarose 5 g SeaKem Gold Agarose 125 g SeaKem Gold Agarose 125 g SeaKem Gold Agarose 25 g SeaKem Gold Agarose 25 g I.D.na Agarose 125 g MetaPhor Agarose 125 g MetaPhor Agarose 125 g MetaPhor Agarose 25 g MetaPhor Agarose 25 g MetaPhor Agarose 500 g MetaPhor Agarose 500 g IsoGel Agarose 25 g IsoGel Agarose 25 g SeaPrep Agarose 25 g DNA Ladder 20 bp Extended DNA Ladder bp DNA Ladder 100 bp Extended DNA Ladder bp SimplyLoad 20 bp Extended SimplyLoad 100 bp DNA SimplyLoad 100 bp Extended SimplyLoad 500 bp DNA DNA Ladder - 20 bp SimplyLoad 20 bp DNA SimplyLoad Tandem DNA DNA QuantLadder Reverse DNA QuantLadder SimplyLoad DNA Quant SimplyLoad DNA Reverse...37 Cat. No. Description Page Number Lambda DNA Ladders S. cerevisiae DNA Stds DNA Marker bp FlashGel Loading dye FlashGel Loading dye FlashGel Loading dye FlashGel Loading dye DNA Marker 1-10 kb FlashGel DNA Marker FlashGel Quant Ladder FlashGel Quant Ladder SYBR Green I Stain μl SYBR Green I Stain μl SYBR Green II Stain μl SYBR Green II Stain μl SYBR Green II Stain μl SYBR Green II Stain μl SYBR Green II Filter (3 in square) 1 ea SYBR Green II Filter (3 in square) 1 ea GelStar Gel Stain GelStar Gel Stain GelStar Gel Stain GelStar Gel Stain GelStar Gel Stain GelStar Gel Stain GelStar Photo Filter GelStar Photo Filter GelStar Photo Filter GelStar Photo Filter GelStar Photo Filter GelStar Photo Filter SYPRO Stain Photo Filter (3 in square) 1 ea SYPRO Red Gel Stain 10 X 50 μl SYPRO Red Gel Stain 500 μl ProSieve Protein Marker 500 μl ProSieve Color Ptn Marker 500 μl ProSieve Color Ptn Marker 100 μl SYPRO Tangerine Gel Stain 5,000X (500 μl) Glyoxal Buffer 1.7 ml SYPRO Ruby Gel Stain 1 L SYPRO Ruby Gel Stain 5 L SYPRO Ruby Gel Stain 200 ml SYPRO Ruby Blot Stain 200 ml Formaldehyde Sample Buffer Formaldehyde Sample Buffer Formaldehyde Sample Buffer Formaldehyde Sample Buffer RNA Marker (0.5 to 9 kb) FlashGel RNA Marker Long Ranger 50% Gel Soln 250 ml Long Ranger 50% Gel Soln 1 L ProSieve 50 Gel Solution 125 ml ProSieve 50 Gel Solution 250 ml MDE Gel Solution 250 ml MDE Heteroduplex Kit 250 ml Heteroduplex Control DNA DNA Marker bp Triple Dye Loading Buffer...41, Gel Slick Solution 250 ml SSCP Stop 5 x 1 ml DNA Loading Buffer , 29, Long Ranger Singel Pk Long Ranger Singel Pk Long Ranger Singel Pk For Sourcebook References see pages 66 & 67

221 Numerical Index Continued Cat. No. Description Page Number Cat. No. Description Page Number Long Ranger Singel Pk Reliant Gels 1% AccuGENE 5X TBE Buffer 10 L...27, 29, Reliant Gels 2% AccuGENE 5X TBE Buffer 20 L...27, 29, Reliant Gels 1% AccuGENE 10X TBE Buffer 10 L...27, 29, Reliant Gels 1% AccuGENE 10X TBE Buffer 20 L...27, 29, Reliant Gels 4% AccuGENE 5X TBE Buffer 4 L...27, 29, Reliant Gels 2% AccuGENE 10X TBE Buffer 4 L...27, 29, Reliant Gels 1% AccuGENE 10X TAE Buffer 4 L...27, 29, Reliant Gels 1% AccuGENE 10X TBE Buffer 1 L... 27, 29, 41, Reliant Gels 1% AccuGENE 10X TAE Buffer 1 L...27, 29, Reliant Gels 1.25% AccuGENE 10X CE Buffer 4 L Reliant Gels 4% AccuGENE 10X CE Buffer 25 ml Reliant Gels 4% AccuGENE 10X MOPS Buffer 1 L...33, Reliant Gels 4% AccuGENE 10X Tris-Glycine 1 L...41, Reliant Gels 4% AccuGENE 10X Tris-Gly SDS 1 L...41, Reliant Gels 2% AccuGENE 10X Tris-Glycine 4 L...41, Reliant Gels 2% AccuGENE 10X Tris-Glycine 1 L...41, Reliant Gels 4% AccuGENE Water, MolBio 1 L...25, Reliant Gel System Tray AccuGENE 0.5M EDTA 100 ml Reliant Gel System Tray AccuGENE 5M NaCl 1 L Reliant Gels 1.25% AccuGENE 20X SSC 1 L Reliant RNA Analysis Kit AccuGENE 10% SDS 100 ml Reliant RNA Analysis Kit AccuGENE 10% SDS 500 ml Reliant RNA Analysis Kit AccuGENE 20X SSPE 1 L Reliant RNA Analysis Kit AccuGENE 50X TAE Buffer 1 L...27, 29, IsoGel Blotting Paper AccuGENE LB Broth 500 ml IEF Plate Accessory Pack AccuGENE Super Broth 500 ml IEF Plate Accessory Pack AccuGENE Super Broth 1 L IsoGel Agarose IEF Plates AccuGENE Water, MolBio 10 L IsoGel Agarose IEF Plates AccuGENE Water, MolBio 20 L IsoGel Agarose IEF Plates AccuGENE 1X PBS 1 L TruBand Gel Anchor AccuGENE 10X PBS 1 L TruBand Gel Anchor AccuGENE Neutralize Solution 1 L Latitude Midigel Chamber AccuGENE Neutralize Solution 10 L TruBand Gel Anchor AccuGENE 20X SSC 20 L TruBand Gel Anchor AccuGENE 20X SSC 10 L FlashGel DNA Cassettes AccuGENE 0.5M EDTA 1 L FlashGel Starter Pack RNA AccuGENE 1X TE Buffer 500 ml FlashGel Dock AccuGENE 1M TRIS-HCl 1 L FlashGel Starter Pack DNA AccuGENE 1M TRIS-HCl 1 L FlashGel RNA Cassettes AccuGENE 1M TRIS-HCl 1 L FlashGel RNA Cassettes AccuGENE 1X TE Buffer 10 L FlashGel DNA Cassettes AccuGENE Mol. Bio. Water 4 L FlashGel DNA Cassettes AccuGENE 20X SSPE 10 L FlashGel DNA Cassettes GelBond Film FlashGel DNA marker GelBond Film FlashGel DNA marker GelBond Film FlashGel Camera GelBond Film FlashGel Recovery Kit GelBond Film FlashGel Recovery Cassettes GelBond Film FlasHGel Recovery Buffer GelBond Film FlashGel System GelBond Film Latitude Gel 1% GelBond Film Latitude HT (24 14 cm) 1% GelBond Film Latitude HT (24 14 cm) 2% GelBond Film Latitude Gel 1% GelBond PAG Film...42, Latitude Gel 2% GelBond PAG Film...42, Latitude HT (24 14 cm) 1% GelBond PAG Film...42, Latitude (10 15 cm) 1% GelBond PAG Film...42, Latitude HT (24 14 cm) 1% GelBond PAG Film...42, Latitude HT (24 14 cm) 4% GelBond PAG Film...42, Latitude HT (24 14 cm) 2% GelBond PAG Film...42, Latitude (10 15 cm) 1% GelBond PAG Film...42, Latitude (10 15 cm) 2% Reliant Gels 1% Latitude (10 15 cm) 4% Indices For Sourcebook References see pages 66 &

222 Numerical Index Continued Indices Cat. No. Description Page Number Cat. No. Description Page Number Latitude HT (24 14 cm) 1% PAGEr Gold Gels 10% 16-well Latitude HT (24 14 cm) 4% PAGEr Gold Gels 12% 16-well Latitude HT (24 14 cm) 2% PAGEr Gold Gels 15% 16-well Latitude RNA 1.25% PAGEr Gold Gels 4-20% 16-well Latitude RNA 1.25% PAGEr Gold Gels 10-20% 16-well Latitude HT (24 14 cm) 2% PAGEr Gold Gels 8-16% 16-well Latitude HT (24 14 cm) 4% PAGEr Gold Gels 4-12% 16-well Agarase 100 units...8, 9, PAGEr Gold Gels 8-16% 16-well Agarase 500 units...8, 9, PAGEr Gold Gels 4-12% 16-well PAGEr Gold Scouting Kit 9cm X 10 cm PAGEr Gold Gels 8-16% 16-well PAGEr Gold Gels 7.5% 12-well PAGEr Gold Gels 4-12% 16-well PAGEr Gold Gels 10% 12-well PAGEr Gold TBE Gels 6% 10-well PAGEr Gold Gels 12% 12-well PAGEr Gold TBE Gels 10% 10-well PAGEr Gold Gels 15% 12-well PAGEr Gold TBE Gels 4-20% 10-well PAGEr Gold Gels 4-20% 12-well PAGEr Gold TBE Gels 6% 10-well PAGEr Gold Gels 10-20% 12-well PAGEr Gold TBE Gels 4-20% 12-well PAGEr Gold Gels 7.5% 10-well PAGEr Gold TBE Gels 6% 16-well PAGEr Gold Gels 10% 10-well PAGEr Gold TBE Gels 10% 16-well PAGEr Gold Gels 12% 10-well PAGEr Gold TBE Gels 4-20% 16-well PAGEr Gold Gels 15% 10-well PAGEr Gold Gels 10% 17-well PAGEr Gold Gels 4-20% 10-well PAGEr Gold Gels 15% 17-well PAGEr Gold Gels 10-20% 10-well PAGEr Gold Gels 15% 17-well PAGEr Gold Gels 7.5% 16-well PAGEr Gold Gels 4-20% 17-well PAGEr Gold Gels 10% 16-well PAGEr Gold Gels 10% 17-well PAGEr Gold Gels 12% 16-well PAGEr Gold Gels 15% 17-well PAGEr Gold Gels 15% 16-well PAGEr Gold Gels 4-20% 17-well PAGEr Gold Gels 4-20% 16-well PAGEr Gold Gels 15% 2D-well PAGEr Gold Gels 10-20% 16-well PAGEr Gold Gels 4-20% 2D-well PAGEr Gold Gels 8-16% 10-well PAGEr Gold Gels 8-16% 17-well PAGEr Gold Gels 4-12% 10-well PAGEr Gold Gels 10% 17-well PAGEr Gold Gels 8-16% 12-well PAGEr Gold Gels 8-16% 2D-well PAGEr Gold Gels 8-16% 12-well PAGEr Gel Chamber PAGEr Gold Gels 4-12% 16-well PAGEr Gel Blot Module PAGEr Gold Gels 4-12% 16-well PAGEr Gel Chamber Kit PAGEr Gold TBE Gels 6% 10-well BE08-238Z10 AccuGENE 10X TBE Buffer 10 L PAGEr Gold TBE Gels 10% 10-well...31 BE08-238Z4 AccuGENE 10X TBE Buffer 4 L PAGEr Gold TBE Gels 4-20% 10-well PAGEr Gold TBE Gels 6% 12-well PAGEr Gold TBE Gels 4-20% 12-well PAGEr Gold TBE Gels 10% 16-well PAGEr Gold TBE Gels 4-20% 12-well PAGEr Gold Gels 10% 17-well PAGEr Gold Gels 12% 17-well PAGEr Gold Gels 15% 17-well PAGEr Gold TBE Gels 4-20% 17-well PAGEr Gold Gels 10% 8+1-well PAGEr Gold Gels 15% 8+1-well PAGEr Gold Gels 4-20% 8+1-well PAGEr Gold Gels 8-16% 17-well PAGEr Gold Gels 8-16% 8+1-well PAGE Gold Scouting Kit 9cm X 10 cm PAGEr Gold Gels 7.5% 12-well PAGEr Gold Gels 10% 12-well PAGEr Gold Gels 12% 12-well PAGEr Gold Gels 15% 12-well PAGEr Gold Gels 4-20% 12-well PAGEr Gold Gels 10-20% 12-well PAGEr Gold Gels 7.5% 10-well PAGEr Gold Gels 10% 10-well PAGEr Gold Gels 12% 10-well PAGEr Gold Gels 15% 10-well PAGEr Gold Gels 4-20% 10-well PAGEr Gold Gels 10-20% 10-well PAGEr Gold Gels 7.5% 16-well For Sourcebook References see pages 66 & 67

223 Alphabetical Index 20 bp DNA Ladder bp Extended Range DNA Ladder bp DNA Ladder bp Extended Range DNA Ladder bp DNA Ladder...37 A AccuGENE 0.5 M EDTA Solution...40 AccuGENE 1 M Tris HCl Buffer...40 AccuGENE 1X PBS AccuGENE 1X TE Buffer...40 AccuGENE 3 M Sodium Acetate, ph AccuGENE 5 M Sodium Chloride AccuGENE 5X TBE Buffer....27, 29, 41 AccuGENE 10% SDS AccuGENE 10X CE Buffer AccuGENE 10X MOPS Buffer....33, 41 AccuGENE 10X PBS AccuGENE 10X TAE Buffer....27, 29, 41 AccuGENE 10X TBE Buffer , 29, 41, 44 AccuGENE 10X Tris-Glycine Buffer , 52 AccuGENE 10X Tris-Glycine SDS Buffer... 41, 52 AccuGENE 20X SSC Buffer...40 AccuGENE 20X SSPE Buffer AccuGENE 50X TAE Buffer....27, 29, 41 AccuGENE LB Broth (Luria Bertani Medium) AccuGENE Molecular Biology Water... 25, 40 AccuGENE Neutralization Solution...40 AccuGENE SSCP Stop Solution AccuGENE Super Broth (Terrific Broth)...40 Agarose I.D.na Agarose...15 InCert Agarose and Megabase DNA Standards...13 MetaPhor Agarose...6 Nucleic Acid Separation...8 NuSieve 3:1 Agarose...7 NuSieve GTG Agarose...8 Protein separation SeaKem Gold Agarose...12 SeaKem GTG Agarose...10 SeaKem LE Agarose...5 SeaKem ME Agarose...14 SeaPlaque Agarose...11 SeaPlaque GTG Agarose...9 Agarose for Protein Separation... 58, 59 Analytical specifications...59 IsoGel Agarose...59 MetaPhor Agarose...59 SeaKem Gold Agarose...59 SeaKem HE Agarose...59 SeaKem HEEO Agarose...59 SeaKem HGT Agarose...59 SeaKem HGT(P) Agarose SeaKem ME Agarose...59 SeaPlaque Agarose...59 Agarose Selection Guide... 4 Agarose and Compatible Techniques...4 Choose the Agarose that is Right for You...4 B -Agarase... 8, 9, 11 Buffers...40 D DNA Ladders and Markers... 36, 37 DNA Loading Buffer (6X)...27, 29, 41 DNA Marker 1 10 kb...37 DNA Marker 50 1,000 bp DNA Marker 50 2,500 bp...37 DNA QuantLadder...37 DNA Reverse QuantLadder...37 DNA Sequincing E Electrophoresis Buffers...41 F FlashGel Camera... 24, 25 Camera Specifications...24 FlashGel DNA Cassettes 9/pk...25 FlashGel DNA Marker, 50 bp 1.5 kb...25 FlashGel DNA Marker, 100 bp 3 kb...25 FlashGel DNA Marker, 100 bp 4 kb...25 FlashGel DNA Starter Kit...25 FlashGel Dock...25 FlashGel Loading Dye...25 FlashGel Loading Dye (5X) FlashGel QuantLadder 100 bp (3 ng) 1.5 kb (30 ng)...25 FlashGel QuantLadder, 100 bp (3 ng) 1.5 kb (30 ng)...25 FlashGel Recovery Buffer...25 FlashGel Recovery Cassettes 9/pk...25 FlashGel RNA Cassettes 9/pk FlashGel RNA Marker, 0.5 kb 9 kb FlashGel System...25 FlashGel System for DNA...18, 19 Cassette Specifications...19 FlashGel System for Recovery...20, 21, 25 Cassette Specifications...21 FlashGel System for RNA...22, 23, 25 Specifications FlashGel System for RNA Starter Pack...25 Formaldehyde Sample Buffer...33 G GelBond Film Rolls GelBond Film Sheets GelBond PAG Film Sheets GelBond PAG Gel Support Film Gel Slick Solution...43 GelStar Gel Stain Photographic Filter... 38, 44 GelStar Nucleic Acid Gel Stain...34, 38, 44 Gel Support Films...42 GelBond Film...42 GelBond PAG Film...42 Glyoxal Sample Buffer...33 H Heteroduplex Control DNA I I.D.NA Agarose...15 InCert Agarose...13 IsoGel Agarose... 56, 59 Analytical Specifications...56 IsoGel Agarose and Precast IsoGel Agarose IEF Plates... 56, 57 Precast IsoGel Agarose IEF Plates...57 L Lambda DNA Ladder...13 Latitude HT Gels... 28, 29 Specifications Latitude HT Precast Gel 100 Well...28 Latitude HT Precast Gels 100 Well...28 Latitude HT Precast Gels 200 Well...28 Latitude Midigel 20 Well...30 Latitude Midigel 40 Well...30 Latitude Midigels...30 Specifications Latitude RNA Midigels Long Ranger 50% Gel Solution...43 Long Ranger Singel Pack...43 Indices For Sourcebook References see pages 66 &

224 Indices 216 Alphabetical Index Continued M Markers...36 MDE Mutation Detection Enhancement Gel Solution...44 MDE Mutation Detection Enhancement Heteroduplex Kit...44 MetaPhor Agarose...6, 59 Molecular Biology Buffers...40 N Nucleic Acid Stains...38 NuSieve 3:1 Agarose NuSieve GTG Agarose... 8 P PAGEr Gold Scouting Kit PAGEr Gold TBE Gels 10, 12 & 16 well...31 Specifications PAGEr Gold Tris-Glycine Precast Gels...49 PAGEr Minigel Chamber...50 Specifications PAGEr Precast Gels for Protein Electrophoresis , 49 Chamber Compatibility...48 PAGEr Precast Gel comb formats Selecting the Best Precast Gel...49 PAGEr Gold Scouting Kit...49 PAGEr Gold Tris-Glycine Precast Gels...49 Specifications Precast Gels Precast Gels and Related Products for RNA Analysis AccuGENE 10X MOPS Buffer Formaldehyde Sample Buffer...33 GelStar Gel Stain Photographic Filter GelStar Nucleic Acid Gel Stain Glyoxal Sample Buffer...33 Reliant and Latitude Precast RNA Gels...32 RNA Marker kb SYBR Green Gel Stain Photographic Filter...34 SYBR Green II Nucleic Acid Gel Stain...34 Precast Gels for DNA and RNA FlashGel Camera... 24, 25 FlashGel System FlashGel System for DNA...19 FlashGel System for Recovery...20, 21 FlashGel System for RNA....22, 23 FlashGel System Products...25 Selection Guide...17 FlashGel System Latitude HT Gels Latitude Midigels PAGEr Gold TBE Gels Reliant Gel Minigels Precast IsoGel Agarose IEF Plates...57 ProSieve 50 Acrylamide Gel Solution...60 ProSieve Color Protein Markers...51 ProSieve Protein Marker...52 Protein Electrophoresis Protein Markers, Buffers, and Stains AccuGENE 10X Tris-Glycine Buffer AccuGENE 10X Tris-Glycine SDS Buffer...52 ProSieve Color Protein Marker...51 ProSieve Protein Marker...52 SYPRO Protein Gel Stain Photographic Filter SYPRO Protein Gel Stains...53 SYPRO Red Protein Gel Stain...54 SYPRO Ruby Protein Blot Stain SYPRO Ruby Protein Gel Stain...54 SYPRO Tangerine Protein Gel Stain...55 R Reliant and Latitude Precast RNA Gels...32 Latitude RNA Midigels...32 Reliant RNA Analysis Kits Reliant RNA Gel System...32 For Sourcebook References see pages 66 & 67 Reliant Gel System Reusable UV Transparent Tray...27 Reliant Minigels... 26, 27 Reliant Gel 8, 12, 20, 24 well...26 Specifications Reliant RNA Analysis Kits...32 Reliant RNA Gel System RNA Marker kb...33 S Saccharomyces cerevisiae DNA Standard SeaKem Gold Agarose...12, 59 SeaKem GTG Agarose SeaKem HE Agarose...59 SeaKem HEEO Agarose...59 SeaKem HGT Agarose SeaKem HGT(P) Agarose SeaKem LE Agarose... 5 SeaKem ME Agarose...14, 59 SeaPlaque Agarose , 59 SeaPlaque GTG Agarose... 9 SeaPrep Agarose...14 Sequencing and Mutation Detection Products... 43, 44 Long Ranger Sequencing Products...43 Singels instrument compatibility MDE Heteroduplex Kit SimplyLoad 20 bp DNA Ladder...37 SimplyLoad 20 bp Extended Range DNA Ladder SimplyLoad 100 bp DNA Ladder...37 SimplyLoad 100 bp Extended Range DNA Ladder...37 SimplyLoad 500 bp DNA Ladder...37 SimplyLoad DNA Ladders...37 SimplyLoad 20 bp DNA Ladder...37 SimplyLoad 20 bp Extended Range DNA Ladder...37 SimplyLoad 100 bp DNA Ladder...37 SimplyLoad 100 bp Extended Range DNA Ladder...37 SimplyLoad 500 bp DNA Ladder...37 SimplyLoad Tandem DNA Ladder...37 SimplyLoad DNA QuantLadder...37 SimplyLoad DNA Reverse QuantLadder...37 SimplyLoad Quantitation Ladders...37 DNA QuantLadder...37 SimplyLoad DNA QuantLadder...37 SimplyLoad DNA Reverse QuantLadder...37 SimplyLoad Tandem DNA Ladder...37 Singels Instrument Compatibility Stains...38, 53 Standard DNA Ladders bp DNA Ladder bp Extended Range DNA Ladder bp DNA Ladder bp Extended Range DNA Ladder bp DNA Ladder...37 Standard DNA Markers...37 DNA Marker 1 10 kb...37 DNA Marker 50 1,000 bp...37 DNA Marker 50 2,500 bp...37 Standard Quantitation Ladders...37 DNA Reverse QuantLadder...37 SYBR Green Gel Stain Photographic Filter , 39 SYBR Green II Nucleic Acid Gel Stain... 34, 39 SYBR Green I Nucleic Acid Gel Stain...39 SYPRO Protein Gel Stain Photographic Filter SYPRO Protein Gel Stains...53 SYPRO Red Protein Gel Stain...54 SYPRO Ruby Protein Blot Stain...55 SYPRO Ruby Protein Gel Stain...54 SYPRO Tangerine Protein Gel Stain...55 T Triple Dye Loading Buffer (6X) Triple-Dye Loading Buffer (6X)...41 TruBand Gel Anchors...29

225 Notes Notes 217

226 Notes Notes 218

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